CN106244500B - One plant of anti-antimony bacterium NXH2 and its application - Google Patents
One plant of anti-antimony bacterium NXH2 and its application Download PDFInfo
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- CN106244500B CN106244500B CN201610848861.4A CN201610848861A CN106244500B CN 106244500 B CN106244500 B CN 106244500B CN 201610848861 A CN201610848861 A CN 201610848861A CN 106244500 B CN106244500 B CN 106244500B
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Abstract
The invention discloses a kind of anti-antimony bacterium Klebsiella (KlebsiellaSp.), deposit number is CGMCC No.12896.Bacterium high resistance antimony of the present invention, while there is anti-As3+、Cd2+、Cr6+、Hg2Etc. various heavies, and promote the characteristic of plant growth, to promoting local vegetation growth, alleviate plant to be of great significance to the murder by poisoning of heavy metal, heavy metal pollution mining soil it is biological prosthetic in there is wide application potential.
Description
Technical field
The invention belongs to field of environmental biotechnology, and in particular to one plant of anti-antimony bacterium NXH2 and its application.
Background technique
In recent years, with the discharge of waste in the continuous expansion and antimony industry of antimony ore acquisition scale, cause mining area all
The soil on side is by serious pollution.As environmental contaminants, antimony and antimonide have been found to be a kind of with genotoxicity
Substance, and to human body have carcinogenesis (Huang et al., 1998; Takahashi et al., 2002;
Beyersmann et al., 2008).In addition, antimony can also cause, injury of lungs, blood urine, fibroblast are dead, sister's dyeing is single
Body exchange, circulation system disease etc. (Huang et al., 1998).Antimony is by Environmental Protection Agency and council, the European Community
It is classified as preferential pollution administration object and pollutant that the Japanese Environment Room monitors closely.In recent years, the pollution situation of China's antimony is more next
It is more serious, gradually by the extensive concern of various circles of society.Meanwhile antimony ore adopts smelting activity while will lead to the huge sum of money with antimony association
Belong to element such as arsenic, mercury etc. and enter mining area supergene environment, polluted mining area farmland, not only influence crop growth, reduces crops
Quality.Very big destruction is also resulted in the soil in mining area.How research, which repairs mining soil, has become problem urgently to be resolved.
Although antimony has very high toxicity for the mankind, some microorganisms can be in the environment of very high concentrations
Growth, it might even be possible to using this element as energy substance, or by the higher Sb of toxicity (III) be oxidized to toxicity compared with
Weak Sb (V), the microorganism remediation of contaminated soil is theoretical as a result, and recovery technique just comes into being (rising should wait, 2007).It is micro-
Biology can outlet by thallus to antimony, highly toxic Sb(III) the Sb(V for being oxidized to hypotoxicity), microorganism is to antimony
The modes such as methylation, thallus absorption reduce antimony to the toxicity of thallus and environment.Anti- antimony 1971, Lyalthova is reported for the first time
1 plant of antimony oxidizing bacteria, the bacterial strain antimony of trivalent can be oxidized to the antimony of pentavalent for itself provide energy (Lyalthova,
1971).There is the report on a small quantity about anti-antimony microorganism successively later, in short, up to the present oneself anti-antimony microorganism through reporting
Type it is opposite also less, therefore, screen have antimony resistance microorganism be realization antimony pollution reparation important link.
It is few about the screening of anti-antimony microorganism and the correlative study of application aspect report at present, Liu Chengzuo (2012) screening
To one plant of resistance to antimony microorganism, it is identified as Penicillium notatum, resistance to antimony concentration is 600mg/L.Strain in the invention patent is Cray
Primary Salmonella (Klebsiella) in one plant of bacterium, anti-antimony effect be apparently higher than it has been reported that Penicillium notatum.And the Cray primary
Salmonella has the function of plant growth-promoting, can generate IAA, siderophore and acc deaminase, has certain growth-promoting effect to plant；
Simultaneously to As3+、Cd2+、Cr6+、Hg2+Etc. various heavies have very strong resistance, have potential application.
Summary of the invention
It is an object of that present invention to provide the one plant bacterium with high resistance antimony, through 16S rDNA sequencing analysis, as a result form
Learn observation, be initially identified as Klebsiella (KlebsiellaSp.), strain number NXH2, the bacterium have anti-As simultaneously3+、
Cd2+、Cr6+、Hg2Etc. various heavies, and promote plant growth characteristic.
Klebsiella of the invention (KlebsiellaSp.) NXH2 is from Lengshuijiang, Hunan Xikuangshan Ore area
The strain of separation screening in plant rhizosphere soil near smeltery, highest are resistant to the heavy metal that antimony concentration is 3300mg/L
Stress.Bacterial strain was deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 23rd, 2016, letter
(unit address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica are postal by referred to as CGMCC
Coding: 100101), deposit number is CGMCC No.12896, is survived through detection.
After cultivating 18h on LB solid medium, colony characteristics are: milky to canescence, and round, edge is complete,
Diameter about 0.85mm, surface is smooth, center slightly protrusion.Its somatic cells feature are as follows: brevibacterium, Gram's staining are negative, nothing
Gemma.
By the 16S rDNA sequence of the bacterial strain by PCR amplification, the amplified production of 1400bp or so length is obtained, amplification produces
Object carries out sequencing through sequencing company, and column will be sequenced and compare with the sequence progress BLAST in GenBank database, as a result
Show the bacterial strain and Klebsiella (Klebsiella) in each bacterium homology highest, similarity is 98% or more.In conjunction with shape
State feature, cultural characteristic and 16S rDNA sequence analysis, the bacterial strain be determined as Klebsiella (Klebsiella sp.).
The bacterial strain contain various concentration Sb(potassium antimony tartrate) CDM solid medium in cultivate certain time, observe
Its growing state, the results showed that, the anti-Sb of the bacterium is very competent, reaches 3300mg/L to trivalent antimony tolerance.
The invention has the following advantages:
Strain of the invention is divided from the plant rhizosphere soil near smeltery, Lengshuijiang Xikuangshan Ore area, Hunan Province
From the anti-antimony of a plant height (Sb) bacterium filtered out, it is identified should for Klebsiella (KlebsiellaSp.).Bacterial strain of the present invention
In addition to the characteristic with very strong preventing from heavy metal antimony, and the bacterial strain is to As3+、Cd2+、Cr6+、Hg2+Equal heavy metals have very strong
Resistance, especially to As3+Tolerable concentration reach 4000mg/L or more.The bacterium also has the function of plant growth-promoting simultaneously, can produce
Raw IAA, siderophore and acc deaminase these three plant growth-promoting factors, and there is promotion to make the plant growth in Sb contaminated soil
With, such as there is certain facilitation effect to growth of rape.At present about a variety of preventing from heavy metal abilities and growth-promoting of anti-antimony bacterium
Research in terms of effect at home and abroad yet there are no relevant report.Therefore, the highly resistance that heavy metal tolerance is impurely screened from mining area promotees
Raw microorganism is of great significance to the local vegetation growth of promotion, alleviation plant to the murder by poisoning of heavy metal.The bacterium is heavy metal-polluted
The mining soil of dye it is biological prosthetic in have wide application potential.
Detailed description of the invention
Fig. 1 Klebsiella of the present invention (KlebsiellaSp. culture).
Fig. 2 Klebsiella of the present invention (KlebsiellaSp. 16S rDNA gene order).
Specific embodiment
Come that the present invention is furture elucidated below by the detailed description of specific embodiment, but is not to limit of the invention
System, only illustrates.
The separation screening of the anti-antimony bacterium of embodiment 1 and anti-Sb ability
The plant rhizosphere soil sample near smeltery, Lengshuijiang, Hunan Xikuangshan Ore area is acquired, is weighed above-mentioned new
Suitable potassium antimony tartrate ([C is added in fresh pedotheque 100g8H4K2O12Sb2·3(H2O)]), make the final dense of antimony in soil sample
Degree is 1000mg/kg, is put in 28 DEG C of constant incubator enrichment cultures one week.Above-mentioned soil sample 10g is taken to be put in equipped with 10 glass
Pearl simultaneously fills in 0.85% NaCl sterile solution of 90mL, 30 DEG C, shakes 30 min in 180 r min-1 shaking tables, keeps sample abundant
It scatters.0.1mL is taken to be applied on CDM culture medium (CDM culture medium prescription: MgSO4·7H2O 2.0 g, NH41.0 g of Cl,
Na2SO41.0 g, K2HPO40.013 g, CaCl2·2H2O 0.067 g, Na-lactate 5.0 g, 15.0 g of agar add
Distilled water wherein makes antimony in final culture medium using potassium antimony tartrate as stress factors in CDM culture medium to 1000mL, pH 7.2)
Concentration be 50 μM, cultivated one week in 30 DEG C of constant incubators, observe its upgrowth situation daily.Above-mentioned initial gross separation is gone out
Bacterial strain progress further isolates and purifies acquisition pure strain, and the strain after separation is connected on inclined-plane, and 4 DEG C of preservations carry out subsequent
Experiment.
Resulting strain inoculated (LB culture medium prescription: peptone 10g, yeast extract in LB culture medium will be screened
5g, NaCl 10g, 15 g of agar, add distilled water to 1000 mL, pH 7.2), 24 h are activated, 2 mL is taken to be transferred to equipped with 100
In 250 mL triangles of mL LB liquid medium, 30 DEG C, 150 r/min shaken cultivations, with nonvaccinated LB Liquid Culture
Base is control, selects the wavelength of 600nm, the OD value of different incubation time bacterial suspensions is measured with microplate reader
(OD600).Using incubation time as abscissa, OD600 is ordinate, draws the growth curve of bacterium, to understand the growth of bacterial strain
Period further studies bacterial strain to the research suitable growth of the tolerance of the heavy metals such as Sb, As, resistance and growth-promoting effect for the later period
The thallus in period.Sb stock solution is added into CDM culture medium, making the final concentration of the Sb in culture medium is respectively 1,2,4,6,8
With 10 mmol/L and concentrations above, heavy metal concentration is successively improved according to experimental result, the anti-Sb bacterial strain that inclined-plane is saved is in LB
24 h are activated in culture solution, single colonie is isolated and purified out on solid medium, then are transferred to the solid training of the Sb containing respective concentration
It supports on base, observes strain growth situation, the minimum concentration for being able to suppress strain growth is the minimum inhibitory concentration of the bacterial strain
(Minimum inhibitory concentration, MIC)。
By above-mentioned separation screening, several anti-Sb bacteriums are obtained, most of plants of bacterium enter stationary phase, bacterial strain logarithm in 48h
Phase is 6h-36h.Wherein, bacterial strain (number NXH2) growth of the present invention is very fast, and stationary phase, therefore subsequent reality are entered after 36h
The thallus taken Pei Yang for 24 hours is tested to be studied.By measurement Sb to isolated strains minimum inhibitory concentration, find bacterial strain NXH2 to Sb's
Resistance is very high, and minimum inhibitory concentration reaches 28mM, is converted into mass concentration substantially 3300mg/L.
The identification of 2 NXH2 bacterial strain of embodiment
The bacterial strain is carried out to the sequence analysis of morphology, cultural characteristic and 16S rDNA.16S rDNA molecule mirror
Surely follow the steps below: the single colonie of picking bacterium is inoculated in liquid LB culture medium, 30 DEG C, 150r/min
Shaking table shaken cultivation is taking out culture solution for 24 hours, and 5000r/min centrifugation 1min takes supernatant, mentions according to bacterial genomes DNA
It takes kit (offer of Tiangeng biochemical technology Co., Ltd), extracts bacterium colony DNA；Universal primer 27F and 1492R are to extraction
DNA of bacteria 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 ' primers are synthesized by Beijing Bioisystech Co., Ltd, farsighted Boxing section；By PCR
Product carries out sequence, and sequencing result BLAST in NCBI database carries out sequence analysis, and carries out homology ratio
Compared with.
The morphological features of NXH2 bacterial strain are as follows: brevibacterium, Gram's staining are negative, and no gemma, colony characteristics are such as
Under: after cultivating 18h on LB solid medium, colony characteristics are: milky to canescence, and round, edge is complete, and diameter is about
0.85mm, surface is smooth, and center slightly protrusion is shown in Fig. 1.The bacterium 16S rDNA gene order length is 1411 bp, by gene sequence
Column are submitted on Genbank, carry out tetraploid rice, 6.0 Software on Drawing phylogenetic tree of MEGA are then used, thus really
Determine the kind of bacterial strain.The result shows that the sequence and Klebsiella (Klebsiella) in each bacterium 16S rDNA gene sequence
Column similarity highest determines that NXH2 bacterial strain is Cray primary in combination with colony morphology characteristic, thallus microscopic features up to 98% or more
Bordetella (KlebsiellaSp.), 16S rDNA gene order is as shown in Figure 2.The bacterium was deposited on August 23rd, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center is referred to as CGMCC (unit address: court, Beijing
The institute 3 of positive area's North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode: 100101), and deposit number CGMCC
No. 12896。
Embodiment 3: tolerance of the Klebsiella NXH2 to various heavy
It prepares respectively and contains various concentration As3+、Cd2+、Cr6+、Hg2The CDM solid medium of heavy metal, Cd2+、Cr6+、Hg2
Heavy metal concentration is followed successively by 100mg/L, 200mg/L, 400mg/L, 800mg/L, 1200mg/L, 1600mg/ from 100mg/L
L, As3+Concentration from 100mg/L, be followed successively by 100mg/L, 200mg/L, 400mg/L, 800mg/L, 1200mg/L, 1600mg/L,
2000mg/L, 3000 mg/L, 4000 mg/L, the processing of heavy metal is not added as control.By Klebsiella
(Klebsiella sp.) NXH2 strain inoculated to without heavy metal CDM culture medium in activation culture for 24 hours, take 0.1mL to distinguish
Be inoculated in containing variety classes, various concentration heavy metal culture medium in, be placed in 30 DEG C of cultures for 24 hours, observation thallus is containing each
Type, various concentration heavy metal culture medium in growing state, the results are shown in Table 1.As can be seen from the table, NXH2 bacterial strain is to above-mentioned
Several heavy metal species all have stronger tolerance, to As3+、Cd2+、Cr6+、Hg2Tolerable concentration be respectively 4000mg/L,
1200mg/L, 1600ml/L and 800mg/L.
Growing state of the 1 Klebsiella NXH2 of table under different heavy metal concentrations
Note: "+" indicates well-grown；"-" expression is not grown；" +/- " expression can be grown, but undergrowth.
The plant growth-promoting characteristic of 4 Klebsiella NXH2 of embodiment
Microorganism with Plant growth promotion is often through the secretion heteroauxin (Indole-3-acetic into environment
Acid, IAA), generate siderophore and acc deaminase, play the effect for promoting plant growth.It, can be with by following experimental implementation
Verifying Klebsiella NXH2 has plant growth-promoting characteristic.The Function Identification method for generating IAA is as follows: in the spy of culture microorganism
Determine fluid nutrient medium and 0. 5 g/L L-Trp (about 2. 5 mmol/L) high-temperature sterilization afterwards is added, is chosen strain bacterial strain with toothpick
Enter fluid nutrient medium, be placed in constant-temperature shaking incubator and cultivate 24 hours for 30 DEG C, draws 2mL bacterium solution, 2mL is added in 2mL bacterium solution
Salkowski reagent (12g FeCl3, 98% H of 430mL2SO4, 570mL H2O it) develops the color.The bacterium solution of pinkiness is then the positive,
Illustrate that this bacterial strain produces IAA.The Function Identification method for generating siderophore is as follows: preparing CAS and detects culture medium, will divide in super-clean bench
Bacterial strain point from purifying is cultivated in 30 DEG C of incubator on CAS detection culture medium；By observing bacterial secretory siderophore
The size of the orange haloing formed does qualitative screening, and orange haloing is bigger, and it is stronger to illustrate that bacterial strain produces siderophore ability.ACC deamination
Enzyme identification method is as follows: preparing DF culture medium (DF culture medium prescription: KH2PO44 g, Na2HPO4 6 g, MgSO4·7H2O 0.2
G, FeSO4·7H2O 0.2 g, glucose 2g, 2 mL of gluconic acid, citric acid 2 g, (NH4)2SO42 g, distilled water constant volume
To 1 L, pH 7.2), strain to be tested is chosen to cross in solid medium with clean toothpick and is cultivated, is placed in constant incubator,
Culture for 24 hours, then by bacterial strain is chosen strain to be tested into being transferred to nitrogen-free and ADF culture medium containing ACC is (with 3 with clean toothpick
Mmol/LACC replaces the (NH in DF culture medium4)2SO4For only nitrogen source) in, it is placed on culture in 30 DEG C of constant incubators
Observation can be primarily determined with ACC deaminase activity as a result, can grow the bacterial strain of bacterium colony on ADF culture medium afterwards for 24 hours.
It is as follows that CAS detects culture medium preparation method:
Solution a: by 0.012gCAS(chrome azurol S) it is dissolved in 10ml distilled water, it adds containing 10mmol/LHCl 2ml
1mmol/L FeCl3Solution；
Solution b: 0.015gHDTMA(cetyl trimethylammonium bromide is taken) it is dissolved in 8mL distilled water；
Dye solution c: solution a being slowly added in solution b, is shaked gently, so that two solution are uniformly mixed mutually, is obtained
Dye liquor c；
By 10 × MM9 salting liquid: (Na2HPO430g, KH2HPO41.5g, NaCl 2.5 g, NH4Cl 5g, distilled water
500ml) 20mL and piperazine Diethanol sulfonic acid 6.04g addition has in the clean triangular flask of distilled water of 150ml, with 50% after mixing
NaOH adjust pH to 6.8, and agar powder 3.2g is added, and obtain culture medium d.
By dye solution c, culture medium d and 1mmolL-1CaCl2、1mmol·L-1 MgSO4·7H2O, 20% grape
Sugar is sterilized separately (115 DEG C, 20min), after 10% acid hydrolyzed casein filtration sterilization, is all placed in 50 DEG C of water-bath heat preservation for standby use.
Above-mentioned 0.2ml 1mmol/L CaCl is measured respectively24ml, 1mmol/L MgSO4·7H2O 6ml, 10% junket egg
The glucose of casamino acid and 2ml 20% is added in culture medium d and dye liquor c is added along bottle wall again, and it is fixed to get blue to mix well
Property detection culture medium, be then poured into culture dish by every ware 30ml, it is stand-by to be placed in aseptic operating platform.
Pass through above-mentioned experiment, the results showed that Klebsiella NXH2 can generate IAA, siderophore and acc deaminase, tool
There is potential plant growth-promoting function.
Influence of the 5 Klebsiella NXH2 of embodiment to growth of rape
Using the farmland soil in Beijing somewhere as potting media.Soil crosses 20 meshes after air-drying, and water content is measured, to dry soil
Weight 20Kg weighs corresponding wind desiceted soil weight, and the amount for the antimony that should be added is configured to 250mL solution, mixes well, makes with above-mentioned soil
Concentration containing antimony is 20 mg/Kg, 50 mg/Kg, 100 mg/Kg, 250 mg/Kg, 500 mg/Kg in soil, with field capacity
70% is added corresponding deionized water, is placed under the conditions of being protected from light and balances 2 months, it is the processing that antimony concentration is 0mg/kg that antimony, which is not added,
To compare, the soil after balance fills basin, spare.Certain amount full grains, rape seed of uniform size are first chosen before experiment
It is put in 10% hydrogen peroxide solution and impregnates 15min, then use ddH2O is rinsed well, and the seed after disinfection is uniformly sprinkled upon paving
In the culture dish for having three layers of gauze, 2d is cultivated in incubator, and germinating energy close seed is chosen when seed shows money or valuables one carries unintentionally and is seeded in
In above-mentioned difference antimony pollution soil, every basin sows 30, and after planting every basin accesses about 10 with watering can8The bacterium solution of CFU/g culture
10mL, not connect the processing of bacterium as control, each processing sets 3 repetitions.Periodically watering, keeps the illumination of daily 8 h, and plant exists
Plant plant height, root long are measured after growing 14 d under greenhouse.As can be seen from Table 2: in the soil of different antimony concentration, addition gram
Rape plant height is managed everywhere in the primary Salmonella NXH2 of thunder and root long is above the processing for not adding bacterium, illustrates that Klebsiella NXH2 is mitigating
Antimony has potential using value to plant poisoning, promotion plant growth aspect.
Influence of the 2 Klebsiella NXH2 of table to rape plant height and root long
| Sb(mg/kg) | Bacterial strain height is not added | Add bacterial strain height | It is long that mycorhiza is not added | Add mycorhiza long |
| 0 | 12.43 | 13.97 | 3.43 | 7.97 |
| 20 | 13.23 | 15.32 | 4.38 | 8.38 |
| 50 | 13.77 | 16.32 | 4.15 | 7.48 |
| 100 | 13.17 | 14.93 | 3.68 | 6.43 |
| 250 | 12.76 | 13.42 | 3.77 | 5.62 |
| 500 | 11.68 | 12.68 | 3.38 | 6.92 |
Claims (4)
1. one plant of anti-antimony bacterium Klebsiella (KlebsiellaSp.), deposit number is CGMCC No.12896.
2. Klebsiella as described in claim 1 (KlebsiellaSp. it) is repaired in the mining soil biology of heavy metal pollution
Application in multiple.
3. application as claimed in claim 2, it is characterised in that: the heavy metal is Sb3+、As3+、Cd2+、Cr6+Or Hg2+。
4. Klebsiella as described in claim 1 (KlebsiellaSp.) promoting the application in plant growth.
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| CN110106123B (en) * | 2019-05-31 | 2021-09-24 | 河南农业大学 | Klebsiella strain Z3 and application thereof in reduction of hexavalent chromium ions |
| 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 |
| CN112980714B (en) * | 2020-09-14 | 2022-04-05 | 广西大学 | Antimony oxidizing bacterium with high tolerance and strong oxidizing ability and application thereof |
| CN113680811B (en) * | 2021-07-05 | 2023-03-10 | 广西大学 | Method for enhancing Klebsiella aerogenes X antimony oxide and application |
| CN115232765B (en) * | 2022-06-16 | 2023-06-16 | 贵州民族大学 | Klebsiella WH-15 and application thereof |
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