CN105483038B - The aerobic arsenic of one plant of thermophilic fiber Cordycepps methylates bacterium SM-1 and its application - Google Patents
The aerobic arsenic of one plant of thermophilic fiber Cordycepps methylates bacterium SM-1 and its application Download PDFInfo
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- CN105483038B CN105483038B CN201510956127.5A CN201510956127A CN105483038B CN 105483038 B CN105483038 B CN 105483038B CN 201510956127 A CN201510956127 A CN 201510956127A CN 105483038 B CN105483038 B CN 105483038B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
Abstract
The invention discloses one plant of thermophilic fiber Cordycepps aerobic arsenic methylate bacterium SM 1 and its application.Present invention separation, identification have obtained one plant of gramnegative bacterium that there is superpower arsenic to methylate with developability, the bacterial strain is one plant of new genera and species of thermophilic fiber section bacterium (Cytophagaceae), name Arsenicibacter palustris SM 1, China typical culture collection center is deposited on October 15th, 2014, preserving number is：CCTCC M2014457.Compared with prior art, the present invention has the advantages that：One plant of bacterium bacterial strain that there is superpower arsenic to methylate with developability is provided, which can be used for paddy soil arsenic pollution reparation, the final harm for mitigating rice field arsenic pollution.
Description
Technical field
The present invention relates to technical field of environmental microorganism, and in particular to the aerobic arsenic of one plant of thermophilic fiber Cordycepps methylates bacterium
SM-1 and its application.
Background technology
Arsenic (As) is a kind of poisonous and hazardous metalloid element, while is also a kind of fearful carcinogenic substance.The whole world is about
The population for having more than 1,000,000 receives the murder by poisoning of this harmful element.In recent decades, the production activity of the mankind is as dug up mine, changing
Work smelting, burning fossil fuel etc. exacerbate propagation and diffusion of the arsenic in water body, air, soil environment.South east asia, this
It is a using rice as the area of main diet, the situation of rice field arsenic pollution increases severely with day.The arsenic content of paddy soil middle and high concentration is not
Growth and the yield of rice can only be influenced；Can also human body be entered by the cumulative function of rice, long-term consumption crowd is caused to be good for
Health endangers, thus is highly desirable to carry out in these areas the reparation and improvement of effective rice field arsenic pollution.
Traditional physics, the method for chemical remediation soil arsenic pollution include the exchange adsorption of coagulation and filtration, iron compound
Deng although these methods are higher to the removal efficiency of arsenic, due to its cost of use costliness, and being caused to the soil that need to be repaired
Secondary destruction so that they are difficult to be applied on a large scale in actually administering.In comparison, biological restoration has without comparable
The advantage of plan, not only invests less, and in-situ immobilization can be directly carried out, and will not cause secondary pollution, so being considered as mesh
One of preceding most promising soil pollution reparation means.Microorganism remediation method occupies very important position in biological restoration
It puts.The microbial process of current existing rehabilitating soil arsenic pollution is mainly made by adding the microorganism with arsenic oxidative function
The trivalent arsenic [As (III)] of reduction-state is converted into the lower pentavalent arsenic of toxicity [As (V)] in soil, since As (V) can be by soil
A large amount of absorption, thus final realize is reduced in the soil liquid using the purpose of arsenic total amount.However, this method can not
The purpose of purifying arsenic contamination soil is really realized, because the total amount of arsenic does not change in (1) soil；(2) As (V)
Toxicity is still very high；(3) As (V) formed in the long-term waterflooding growth course of rice will be gradual under the action of microorganism
Reduction, and be returned to the stronger As of toxicity (III), thus we be highly desirable to find it is a kind of more effective, more reliable micro-
Biological renovation method removes the arsenic pollution in soil.
Toxicity can be converted into volatilization by the As (III) of reduction-state by being methylated using the arsenic of paddy soil microorganism
Dimethyl arsenic [the DMAs of the organo-arsenic class compound lower than As (the V)-monomethyl arsenic [MMAs (V)] including pentavalent, pentavalent
(V)] and the arsenic trimethide oxide of pentavalent [TMAs (V) O], while arsenic hydride (AsH can be also generated in this process3), diformazan
Arsenic is taken away soil ontology by the escaping gas such as base arsenic [DMAs (III) H] or arsenic trimethide [TMAs (III)], this is transformed
Journey is one of important component of global arsenic cycle, while is also regarded as the biological prosthetic another hope of arsenic in soil.However mesh
The preceding known arsenic from the rice field microorganism that methylates only has two plants, and one plant is Japanese scholars Masato Kuramata deciles
Streptomyces sp.GSRB54 from identification；Another strain is that the identification arsenic such as Wang Peipei methylates one plant of function from rice
The sulfate reducing bacteria Clostridium sp.BXM in field.Although this two plants of bacteriums are proved the methylation capacity all with arsenic,
Researcher has also identified a trivalent arsenic methyl transferase gene (arsM) out of each body, however this two plants of bacterial strains
Do not have the ability of arsenic volatilization, cannot all generate volatile arsenic compound and arsenic be taken away into soil, and the two bacterial strains are not
Carry out corresponding soil incubation experiment to prove its effect in ecological environment of soil system, thus they are difficult by veritably
Improvement applied to paddy soil arsenic pollution.
In previous paddy soil water-saving irrigntion experiment, we are found that a with apparent arsenic first in laboratory conditions
The soil of base ability, the present invention are exactly one plant to be detached and identified from this part of soil there is superpower arsenic to methylate and volatilize
The microbial strains of ability, the bacterial strain can be effectively applied to the improvement of paddy soil arsenic pollution.
Invention content
The purpose of the present invention is to provide one plant there is arsenic to methylate and newly belong to new bacterium with the thermophilic fiber Cordycepps of developability
Arsenicibacter palustris SM-1。
It is another object of the present invention to provide Applied D emonstration of the bacterial strain in the improvement of rice field arsenic pollution.
The purpose of the present invention can be achieved through the following technical solutions：
Present invention separation, identification have obtained one plant of gramnegative bacterium that there is superpower arsenic to methylate with developability,
The bacterial strain is one plant of new genera and species of thermophilic fiber section bacterium (Cytophagaceae), names Arsenicibacter
Palustris SM-1 were deposited in China typical culture collection center on October 8th, 2014, and preserving number is：CCTCC
M2014457。
(1) morphological feature of bacterial strain：
The morphological feature of bacterium, research knot are studied using means such as conventional Gram's staining and electron microscope observation
Fruit shows bacterial strain SM-1 as rod-shaped Gram-negative bacteria, and without flagellum, without motility, the size of cell is 0.77-
0.85×3.8-5μm.In R2After being cultivated 2 days on A culture mediums, colonial morphology is rounded, surface bulge, neat in edge, crocus,
The size (d) of bacterium colony is 1~2mm at this time.
(2) physio-biochemical characteristics of bacterial strain
Bacterial strain SM-1 is not grown under the conditions of supporting well in LB culture mediums；It is grown in enriched medium, but growing way is poor；It should
The optimum medium of bacterium is R2A, should be the result shows that SM-1 be one plant of oligotrophic type bacterium supported well.The temperature that the bacterial strain can be grown
Ranging from 15-45 DEG C, pH 6-8, the salt being resistant to (NaCl) concentration range is 0-0.5%.The oxidase active of the bacterium is the moon
Property, contact enzymatic activity is the positive, is found after being identified with reagent strip ID32GN, and in 32 kinds of different carbon sources, which can only utilize
(D-Glucose, salicin, D- melibioses, N-Acetyl-D-glucosamine, sucrose, maltose, glycogen) 7 kinds therein, carbon source profit
Substrate spectrum is narrow.
(3) 16S rRNA sequences and Phylogenetic Analysis
First using CTAB methods extract bacteria total DNA, using the DNA as template, using universal primer F27 and R1492PCR come
Amplification obtains the PCR product of the 16S rRNA genes of the bacterial strain, the recovery purifying 1500bp or so, itself and carrier PMD18T are connected
Conversion enters in bacillus coli DH 5 alpha after connecing, and selects positive clone molecule and sequencing company is sent to be sequenced, the sequence inputting of survey time
Blast is carried out in NCBI with other 16S rRNA gene orders to compare.Comparison result shows the bacterial strain and thermophilic cellulose Cordycepps
The germy homology of institute is respectively less than 88%, it is believed that is one plant of new genera and species of thermophilic cellulose Cordycepps.With reference to above-mentioned life
Physiological-biochemical characteristic, 16S rRNA sequences as a result, the bacterial strain should belong to thermophilic cellulose Cordycepps, be named as Arsenicibacter
palustris SM-1。
The preserving number is CCTCC NO：The thermophilic fiber section bacterium SM-1 of M2014457 is in trivalent arsenic pollution is administered
Using.
The preserving number is CCTCC NO：The thermophilic fiber section bacterium SM-1 of M2014457 is administering trivalent As polluted soil
In application.
Compared with prior art, the present invention has the advantages that：Providing one plant, there is superpower arsenic to methylate and wave
The bacterium bacterial strain of hair ability, the bacterial strain can be used for paddy soil arsenic pollution reparation, the final harm for mitigating rice field arsenic pollution.
Description of the drawings
Fig. 1：The scanning electron microscopic picture of bacterial strain SM-1.
Fig. 2：The Phylogenetic Analysis of bacterial strain SM-1.
Fig. 3：Bacterial strain SM-1 is to the inversion cuver figure of As (III).(A)R2The metamorphosis of arsenic in A culture mediums；(B) it volatilizees
The changes of contents figure of property arsenic.
Fig. 4：The form and content of arsenic in the soil liquid (A) and volatile matter (B).
Biomaterial preservation information
Arsenicibacter palustris SM-1 are deposited in China typical culture collection center, in preservation address
Wuhan Wuhan University of state, preservation date on October 8th, 2014, preserving number are：CCTCC NO：M2014457.
Specific embodiment：
Embodiment 1：The enrichment of pedotheque and arsenic methylate separation, purifying, the preservation of bacterium SM-1
It fetches and is added in the enriched medium of 100mL from the paddy soil 2g in crossdrift arsenic pollution area, enriched medium
It forms as (g/L)：ST10-1(0.5g peptones, 0.05g yeast extracts), 0.5% glucose, 10 μM of As (III).Culture medium is put
200rpm, 30 DEG C of shaking table culture one week are placed in, takes out 5mL switchings from pregnant solution after a week in new 100mL enrichment cultures
It concentrates, continues to cultivate in same culture conditions, above-mentioned switching process is repeated twice, and 2mL is finally taken out from pregnant solution is used for arsenic
Morphological analysis, then sample crosses 0.22 μM of filter membrane for first to centrifuge 2min with centrifuge 12000rpm before detecting.It adopts
Detect in pregnant solution whether have organo-arsenic with High performance liquid chromatography-inductively coupled plasma mass spectrometry instrument (HPLC-ICP-MS)
Generation of the compound such as DMAs (V), MMAs (V), TMAs (V) O, the mobile phase of detection is 8.5mM (NH4)2HPO4、8.5mM
NH4NO3, pH 6.0, working flow rate is 1.2ml min-1, germanium is designated as in work.Determine that pregnant solution has the function of methylating for arsenic
Afterwards, tablet is positioned in 30 DEG C of incubators and cultivates on enriched medium solid plate by dilution spread, flat from these after 5 days
Picking single bacterium colony is isolated and purified on plate, separately verifies the methylation activity of the arsenic of these bacterial strains.It is preserved and determined with glycerol tube
Bacterial strain with arsenic methylation activity, final concentration of the 20% of glycerine used, glycerol tube is finally preserved in -70 DEG C of refrigerator
In.
Embodiment 2：The identification of strain
(1) measure of Physiology and biochemistry property：
The physio-biochemical characteristics of 1. bacterial strain SM-1 of table
Note：+ represent to utilize；Expression cannot utilize
(2) molecular biology identification
Bacteria total DNA is extracted using CTAB methods, using the DNA as template, is expanded using universal primer F27 and R1492PCR
The PCR product of the 16S rRNA genes of the bacterial strain, the recovery purifying 1500bp or so are obtained, after it is connect with carrier PMD18T
Conversion enters in bacillus coli DH 5 alpha, selects positive clone molecule and sequencing company is sent to be sequenced, in the sequence inputting NCBI of survey time
Blast is carried out with other 16S rRNA gene orders to compare.The sequence has been committed to NCBI GenBank, and number of registration is
KT989310.With reference to above-mentioned physio-biochemical characteristics, 16S rRNA sequences as a result, the bacterial strain should belong to thermophilic CELLULOLYTIC BACTERIUM
Section is named as Arsenicibacter palustris SM-1.
Embodiment 3：Bacterial strain arsenic methylates the detection with developability under laboratory condition
R2The component of A culture mediums is (g/L):Dusty yeast 0.5g, tryptone 0.5g, casamino acid 0.5g, grape
Sugared 0.5g, soluble starch 0.5g, dipotassium hydrogen phosphate 0.3g, epsom salt 0.05g, Sodium Pyruvate 0.3g.By bacterial strain SM-1
Be inoculated in 100mL aforesaid liquid culture mediums, 37 DEG C, 200rpm shaking table culture diels make the OD of thalline600Value reaches 1.0, this
When the seed liquor of 1mL taken out from seed culture medium add in the R that new 100mL adds 10 μM of As (III)2A fluid nutrient mediums
In, processing sets three repetitions respectively, and switching liquid continues to cultivate, be taken once every 4 hours under above-mentioned same culture conditions
Solution example, after the solution 12000rpm centrifugations 2min of taking-up, supernatant crosses 0.22 μM of filter membrane, is stored in 4 degree of refrigerators accurate
It is ready for use on machine and measures arsenic content and form.10%AgNO has been impregnated in use while solution example is taken3Silicon particle collect
The volatility arsonium compound escaped from culture medium collects the concrete mode and subsequent sample of volatility arsine using gas device
Pickling, which takes off, can refer to my previous research work (Ke Huang et al.Genetically Engineering
Bacillus subtilis with a Heat-Resistant Arsenite Methyltransferase for
Bioremediation of Arsenic-Contaminated Organic
Waste.Appl.Environ.Microbiol.2015,81(19),6718-6724.).Result of the test is as shown in figure 3, from figure
We can see that bacterial strain SM-1 methylates with superpower arsenic and developability, the concentration of As (III) is with culture in solution
The extension of time and reduce, by culture for 24 hours, bacterial strain SM-1 can not only convert As (III) almost all of in solution
The DMAs (V) extremely low for 2.8 μM of toxicity and 2.3 μM of almost non-toxic TMAs (V) O, while can also be by approximately half of As
(III) (48%) in the form of 36 μ g TMAs (III) volatile zone from fluid nutrient medium.
Embodiment 4：Bacterial strain SM-1 is to the repairing effect of crossdrift As polluted soil
By 100g As polluted soils, (total arsenic concentration is about 25mg L-1) and 100mL deionized waters be placed in the triangular flask of 100mL
Middle mixing, experiment process set two groups, and one group is to access the R that 20mL sterilization treatments are crossed2A fluid nutrient mediums；Another group is then accessed
SM-1 bacteria suspensions fresh 20mL, two groups of processing are placed in cultivating in 28 DEG C 200 turns of shaking table 5 days, every group of experimental setup four
It repeats.After culture, the mixed liquor of 5mL is taken out from every bottle of Soil slurry liquid with the liquid-transfering gun of 5mL, by these mixed liquors
13,000rpm carry out centrifugal treating, and Aspirate supernatant crosses HPLC-ICP-MS on 0.22 μm of filter membrane and detects arsenic content and shape therein
State.Consistent with the fluid nutrient medium culture bacterial strain is：We also had collected in the last moment that current soil remediation is tested from
The volatility arsine to escape in experiment soil, these volatile arsenic equally can be analyzed and be determined with HPLC-ICP-MS
Amount.Result of the test is as shown in Figure 4.As can be seen from Figure 4, only have minimal amount of organo-arsenic [15.6 μ g/L in the soil liquid for not connecing bacterium
DMAs (V)] it generates, most arsenic morphology is still based on inorganic arsenic in solution.However, the DMAs in the soil for connecing bacterium
(V) content significantly rises, and reaches 245.7 μ g/L, while we also detect 13.1 μ g/L TMAs (V) in the soil liquid
O, with not plus compared with the soil of bacterium, the amount of inorganic trivalent arsenic in the soil of bacterium being added to be substantially reduced, while add in bacterial strain SM-1 also to make
It obtains soil and produces a small amount of volatile arsenic [9.9ng TMAs (III)], so as to reduce the concentration of total Arsenic In Soil.This reality
It tests and shows that bacterial strain SM-1 can significantly reduce the content of hypertoxic inorganic arsenic with useful effect in the paddy soil of arsenic pollution,
Generate the relatively low organo-arsenic of toxicity and volatile arsenic.
Claims (3)
1. one plant has the thermophilic fiber section bacterium SM-1 that arsenic methylates with developability, it is characterised in that：October 15 in 2014
Day is deposited in China typical culture collection center, and preserving number is：CCTCC NO：M2014457.
2. applications of the thermophilic fiber section bacterium SM-1 described in claim 1 in trivalent arsenic pollution is administered.
3. application according to claim 2, it is characterised in that：Thermophilic fiber section bacterium SM-1 described in claim 1 is being controlled
Manage the application in trivalent As polluted soil.
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| CN112680464B (en) * | 2020-12-10 | 2022-04-26 | 南京农业大学 | Monomethylarsenic and trivalent antimony oxidase gene arsV, and protein coded by same and application thereof |
| CN114410553B (en) * | 2022-03-29 | 2022-06-14 | 广东省科学院生态环境与土壤研究所 | Bacillus with aerobic arsenic methylation and volatilization functions and application thereof |
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| CN103834589A (en) * | 2014-01-13 | 2014-06-04 | 福建农林大学 | Achromobacter xylosoxidans and application thereof |
| CN104004691A (en) * | 2014-06-16 | 2014-08-27 | 中南大学 | Bacterial strain used for arsenic polluted soil remediation and application method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834589A (en) * | 2014-01-13 | 2014-06-04 | 福建农林大学 | Achromobacter xylosoxidans and application thereof |
| CN104004691A (en) * | 2014-06-16 | 2014-08-27 | 中南大学 | Bacterial strain used for arsenic polluted soil remediation and application method thereof |
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| Mechanisms of arsenic biotransformation;Marie Vahter;《Toxicology》;20021231;全文 * |
| Microbial Arsenic Methylation in Soil and Rice Rhizosphere;Yan Jia et al.;《Environ. Sci. Technol.》;20131231;第43卷;全文 * |
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