CN105985923A - High-efficiency anaerobic arsenic-reducing bacterium strain - Google Patents
High-efficiency anaerobic arsenic-reducing bacterium strain Download PDFInfo
- Publication number
- CN105985923A CN105985923A CN201610251435.2A CN201610251435A CN105985923A CN 105985923 A CN105985923 A CN 105985923A CN 201610251435 A CN201610251435 A CN 201610251435A CN 105985923 A CN105985923 A CN 105985923A
- Authority
- CN
- China
- Prior art keywords
- arsenic
- strain
- alkaliphilus
- reducing bacteria
- imb
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a high-efficiency anaerobic arsenic-reducing bacterium strain, belonging to the field of environmental microorganisms. The 16S rDNA sequence analysis by DNA-DNA hybridization identification indicates that the strain is an Alkaliphilus new species and is named Alkaliphilus sp.IMB, and the collection number is CGMCC 1.5191. The optimal pH range for the strain growth is 9.0, the optimal salt concentration is 10-15 g.L<-1>, the minimal inhibitory concentration for As (III) is 5mM, and the minimal inhibitory concentration for As (V) is 40mM. Under anaerobic conditions, the stain can reduce more than 80% of As (V) within 120 hours. The strain has the structural gene arrA of an anaerobic arsenic reductase; and the synchrotron radiation research detects that the arsenic reduction occurs in cytomembranes.
Description
Technical field
The present invention relates to a strain efficient anaerobic arsenic reducing bacteria, belong to field of environment microorganism, this bacterial strain can be at anaerobic condition
The lower arsenic reductase Arr albumen utilized on cell membrane carries out the efficient reduction of arsenic.
Background technology
Arsenic is a kind of teratogenesis carcinogen being widely present in nature, main with trivalent and pentavalent shape in natural water
Formula exists.Trivalent arsenic has stronger animal migration, and toxicity is 25-60 times of pentavalent arsenic.Natural environment evolves some permissible
The microorganism of anti-arsenic, wherein arsenic reducing bacteria is the main groups of arsenic resistant microorganism, including aerobic arsenic reducing bacteria and anaerobism arsenic also
Former bacterium.The reduction mechanism of aerobic arsenic reducing bacteria is can to synthesize ArsC reductase in its kytoplasm, and pentavalent arsenic is reduced to trivalent arsenic；
The reduction mechanism of anaerobism arsenic reducing bacteria is to synthesize Arr reductase on its cell membrane, and pentavalent arsenic can be reduced to trivalent by this enzyme
Arsenic.Arsenic also pathogenic microorganism has played very important effect in the arsenic geochemical cycle of nature.Current research mainly collects
In the geochemical cycle of arsenic that participates in aerobic microbiological, study starting evening about the reduction of arsenic anaerobe, study base
Plinth is weaker.
The present invention from the Hetao Plain Soils In The Region of Inner Mongol screening and separating to a strain efficient anaerobic arsenic reducing bacteria, this bacterial strain pair
Arsenic Transport And Transformation in the anaerobic environments such as research this area natural water has important directive significance.
Summary of the invention
The purpose of present invention is to provide a strain efficient anaerobic arsenic reduction bacterial strain.
According to a specific embodiment of the present invention, separation screening is to a strain efficient anaerobic arsenic reduction bacterial strain, identified
A novel species strains A lkaliphilus sp.IMB for basophilic Pseudomonas.The depositary institution of this bacterial strain is Chinese microorganism strain
Preservation committee common micro-organisms center, (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Chinese Academy of Sciences's microorganism
Institute, postcode 100101), deposit number is CGMCC 1.5191.
According to a specific embodiment of the present invention, efficient anaerobic arsenic reduction bacterial strain Alkaliphilus sp.IMB shape
Looks are characterised by: shaft-like, terminal spore, Gram-positive.Growth optimum pH scope is 9.0, and the suitableeest salinity is 10-15g
L-1。
According to a specific embodiment of the present invention, the arsenic resistant properties of Alkaliphilus sp.IMB is: trivalent arsenic
Minimum growth inhibitory concentration is 5mM, and pentavalent arsenic minimal inhibitory concentration is 40mM.Under anaerobic, 120h reducible 80% with
On As (V).
According to a specific embodiment of the present invention, the anti-mechanism of arsenic of Alkaliphilus sp.IMB is: pentavalent arsenic quilt
Structural gene arrA of anaerobism arsenic reductase is reduced to trivalent arsenic, and Synchrotron Radiation Study finds that arsenic reduction occurs on cell membrane.
Accompanying drawing explanation
Accompanying drawing 1 is the Phylogenetic figure of the efficient anaerobic arsenic reducing bacteria Alkaliphilus sp.IMB of the present invention.From
It can be seen that this bacterial strain is the bacterial strain of Alkaliphilus in figure.
Accompanying drawing 2 is the morphological characteristic figure of the efficient anaerobic arsenic reducing bacteria Alkaliphilus sp.IMB of the present invention.From figure
It can be seen that it is shaft-like, terminal spore.
Accompanying drawing 3 is growth pH and the spy of salinity of the efficient anaerobic arsenic reducing bacteria Alkaliphilus sp.IMB of the present invention
Levy figure.It can be seen that the most suitable growth pH of this bacterium is 9, the most suitable growth salinity is 10-15g L-1。
Accompanying drawing 4 is the pentavalent arsenic reduction kinetics of the efficient anaerobic arsenic reducing bacteria Alkaliphilus sp.IMB of the present invention
And growth curve characteristic pattern.It can be seen that pentavalent arsenic is reduced from the beginning of the exponential phase of cell by bacterial strain, permissible
The pentavalent arsenic of reduction more than 80% in 120h.
Accompanying drawing 5 is the efficient anaerobic arsenic reducing bacteria Alkaliphilus sp.IMB arsenic reductase ArrA albumen of the present invention
ArrA gene electrophoretogram, wherein M:ladder；A: positive control strain PCR expands the ArrA structural gene arrA band obtained；
B:Alkaliphilus sp.IMB expands the arrA band obtained.
Accompanying drawing 6 coerces the synchrotron radiation spectrum analysis knot of Alkaliphilus sp.IMB in ligand system for 1mL pentavalent arsenic
Really.Wherein A:As (III) and the As L of As (V) standard specimenIIILimit NEXAFS collection of illustrative plates；B and C: be respectively As (III) and As (V) carefully
The scanning transmission X-ray micro-image (the shaft-like and spherical material of black represents cell) that born of the same parents are distributed about.
Detailed description of the invention
Embodiment 1 efficient anaerobic arsenic reducing bacteria Alkaliphilus sp.IMB is isolated and purified, identify and preservation
(1) separation of bacterial strain
Being joined by 1g pedotheque in 10mL CDM culture medium, under the conditions of 30 DEG C, lucifuge cultivates 72h, all of operation
All carry out in anaerobism glove box.Every 72h takes the culture fluid of 1% and is transferred in fresh CDM culture medium, after being so repeated 3 times,
Take 1mL culture fluid and cross the filter membrane of 0.22 μm, measure valence state and the content of arsenic.Select there is the flora of arsenic reducing power as target
Flora, utilizes Hunter rolling tube technique to carry out isolated and purified obtaining Alkaliphilus sp.IMB.The component of CDM culture medium:
0.225g K2HPO4；0.225g KH2PO4；0.117g MgSO4·7H2O；0.46g NaCl；0.225g(NH4)2SO4；0.424g
Na3AsO4；4.2g NaHCO3；2.24g sodium lactate；1.0g yeast powder；Trace element mixed liquor 10mL, vitamin solution 10mL；
1L H2O, 13g agar.
(2) extraction of bacteria total DNA
From solid plate, picking list colony inoculation is in 20mL CDM fluid medium, 30 DEG C of lucifuge Anaerobic culturel 72h.4
DEG C 12000r/min is centrifuged 5min and collects thalline, after 1mL TE washing thalline twice, adds 1mL TE and is fully suspended by thalline,
Add lysozyme 0.001g mixing, place to liquid clarification in 37 DEG C of water-baths, till fragment is arranged at bottom.5 μ L are added in liquid
RNase (10mg/mL), 37 DEG C of water-bath 30min, add 10%SDS 200 μ L, 60～65 DEG C process to liquid clarify.Add
E.C. 3.4.21.64 powder is a little, and 37 DEG C process 1～2 hour, then add 1/5 volume 5M NaClO in liquid4.Use equal-volume phenol:
Chloroform: isoamyl alcohol extraction 1～2 times, 12000r/min is centrifuged 15min and collects supernatant, uses equal-volume chloroform: isoamyl alcohol extraction 1～2
Secondary, 12000r/min is centrifuged 10min and collects supernatant.In supernatant, add 1/10 volume 3M NaAc, 2 times of volume dehydrated alcohol in-
20 DEG C of precipitation 30min, choose the DNA being settled out with Glass rod, add overnight to process in 70% ethanol and desalt.After finally drying
DNA be dissolved in 400mL ddH2In O ,-20 DEG C of preservations are stand-by.
(3) amplification of 16S rDNA
With procaryotic type strain colibacillary 16S rDNA primers, as shown in table 1:
The primer of table 1 16S rRNA gene
With extraction genomic DNA as template, PCR expands 16S rDNA sequence, and amplification system and amplification program are as follows:
Amplification system
The amplification program of 16S rDNA:
Obtain the band of about 1.5Kb, carried out this band reclaiming purification, and deliver order-checking.Sequencing result is carried out
The BLAST comparison of line, and utilize the Phylogenetic of Mage5 software building bacterial strain, as shown in Figure 1, this bacterial strain is result
Basophilic Pseudomonas bacterial strain.It is general that this strains A lkaliphilus sp.IMB has been preserved in China Committee for Culture Collection of Microorganisms
CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode (are called for short in logical microorganism center
100101), preserving number CGMCC 1.5191.
The research of embodiment 2 anaerobism arsenic reducing bacteria Alkaliphilus sp.IMB growth characteristics
(1) bacterial strain pattern
Alkaliphilus sp.IMB is cultivated to logarithmic (log) phase, collects thalline.The penta of 2.5% configured with 0.1M PBS
Bacteria samples is fixed 2h by dialdehyde solution.Sample is immersed in 2h in 1% osmic acid, uses ethanol serial dehydration, be embedded in resin
In (Epon 812, Fenwal, US).Ultramicrotome cuts out the section of 70nm, is dragged on copper mesh, with acetic acid uranium and Fructus Citri Limoniae
After lead plumbate dyes, use tem observation.
(2) strain growth pH
Alkaliphilus sp.IMB is cultivated logarithmic (log) phase.Inoculum concentration according to 1% is inoculated into the new of different pH respectively
In fresh CDM culture medium (pH 6, pH 7, pH 8, pH 9, pH 10, pH11, pH12, pH 13), survey an OD every 6h600, note
Record and draw time dependent OD600Curve.
(3) strain growth salinity growth
Alkaliphilus sp.IMB is cultivated logarithmic (log) phase.Inoculum concentration according to 1% is inoculated into different sodium chloride respectively
In the fresh CDM culture medium of concentration (0,10,20,30,40,50,60,70,80g/L), survey an OD every 6h600, record and paint
Make time dependent OD600Curve.
Embodiment 3 anaerobism arsenic reducing bacteria Alkaliphilus sp.IMB anaerobism arsenic reduction feature
(1) minimum growth inhibitory concentration (MIC)
Alkaliphilus sp.IMB is cultivated logarithmic (log) phase.Inoculum concentration according to 1% is inoculated into different As respectively
(III) concentration (0-20mM) and As (III) concentration (0-100mM), measures the OD of bacterium solution after 30 DEG C of Anaerobic culturel 72h of lucifuge600
Value.
(2) research of arsenic reduction characteristic
Alkaliphilus sp.IMB is cultivated logarithmic (log) phase.Inoculum concentration according to 1% is inoculated into respectively containing 1mol
L-1In the CDM fluid medium of As (V).Every 2-4h, take suspension sample and cross 0.22 μm filter membrane, in monitoring 0-60h, arsenic morphology and
The change procedure of concentration.Ultraviolet spectrophotometer (HACH 2800, the U.S.) is used to measure the OD600 value of bacterium solution.Different shape arsenic
Concentration measures and uses high performance liquid chromatography-atomic fluorescence combination method, HPLS-AFS instrument (Ji Tian, China) to measure.
Embodiment 4 anaerobism arsenic reducing bacteria Alkaliphilus sp.IMB arsenic reduction mechanism
(1) anaerobism arsenic also protogene arrA amplification
With extraction genomic DNA as template, the degenerate primer of the arrA gene of report is shown in Table 2, and PCR expands arrA gene sequence
Row (amplification system and amplification program see below).Obtaining the band of about 279bp, electrophoresis 20min under the conditions of 160V, result is shown in accompanying drawing
5。
The primer of table 2 arrA gene
Amplification system
The amplification program of 16S rDNA:
(2) determination of anaerobism arsenic also original position
Alkaliphilus sp.IMB is cultivated logarithmic (log) phase, collects thalline.The penta of 2.5% configured with 0.1M PBS
Bacteria samples is fixed 2h by dialdehyde solution.Sample is immersed in 2h in 1% osmic acid, uses ethanol serial dehydration, be embedded in resin
In (Epon 812, Fenwal, US).Ultramicrotome cuts out the section of 500nm, is fixed on specimen holder.Select
1333.3,1339.4 and 1342.6eV tri-energy points, scan XY direction, it is thus achieved that the figure in this region in the sample area of 3 × 4 μm
Picture.The dual intensity software utilizing line station to provide carrys out analytical data, it is thus achieved that the spatial distribution image (Fig. 6) of different valence state arsenic.Standard sample
Product As (V) and the ENXAS spectrogram of As (III), obtain (accompanying drawing 6) by total electron yield (TEY) pattern.This test is same in Shanghai
Software X-ray beam line station (BL08U) of step radiating light source (SSRF) completes.
Claims (5)
1. a strain efficient anaerobic arsenic reducing bacteria, it is characterised in that: pantoea agglomerans belongs to bacterial strain, named Alkaliphilus
sp.IMB。
A strain efficient anaerobic arsenic reducing bacteria the most as claimed in claim 1, it is characterised in that: shaft-like, terminal spore, gram sun
Property bacterium, this strain growth optimum pH scope is 9.0, and the suitableeest salinity is 10-15g L-1。
A strain efficient anaerobic arsenic reducing bacteria the most as claimed in claim 1, it is characterised in that: trivalent arsenic minimum growth inhibitory concentration
For 5mmol L-1, pentavalent arsenic minimal inhibitory concentration is 40mmol L-1, under anaerobic, 120h reducible more than 80%
As(V)。
A strain efficient anaerobic arsenic reducing bacteria the most as claimed in claim 1, it is characterised in that: this bacterial strain has has anaerobism arsenic to reduce
Structural gene arrA of enzyme, arsenic reduction occurs on cell membrane.
A strain efficient anaerobic arsenic reducing bacteria the most as claimed in claim 1, its deposit number is CGMCC 1.5191.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610251435.2A CN105985923A (en) | 2016-04-21 | 2016-04-21 | High-efficiency anaerobic arsenic-reducing bacterium strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610251435.2A CN105985923A (en) | 2016-04-21 | 2016-04-21 | High-efficiency anaerobic arsenic-reducing bacterium strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105985923A true CN105985923A (en) | 2016-10-05 |
Family
ID=57043955
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610251435.2A Pending CN105985923A (en) | 2016-04-21 | 2016-04-21 | High-efficiency anaerobic arsenic-reducing bacterium strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105985923A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110467274A (en) * | 2019-08-27 | 2019-11-19 | 广东省生物工程研究所（广州甘蔗糖业研究所） | The new opplication of acidovorax facilis LS-1 and kit and its application for repairing arsenic pollution |
-
2016
- 2016-04-21 CN CN201610251435.2A patent/CN105985923A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110467274A (en) * | 2019-08-27 | 2019-11-19 | 广东省生物工程研究所（广州甘蔗糖业研究所） | The new opplication of acidovorax facilis LS-1 and kit and its application for repairing arsenic pollution |
| CN110467274B (en) * | 2019-08-27 | 2022-03-22 | 广东省生物工程研究所（广州甘蔗糖业研究所） | Novel application of acidovorax LS-1, kit for repairing arsenic pollution and application of kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lee et al. | Spirosoma radiotolerans sp. nov., a gamma-radiation-resistant bacterium isolated from gamma ray-irradiated soil | |
| Kajale et al. | Cultivation of diverse microorganisms from hypersaline lake and impact of delay in sample processing on cell viability | |
| CN105985923A (en) | High-efficiency anaerobic arsenic-reducing bacterium strain | |
| CN104396551A (en) | Method for ecological restoration of soil in rare earth mining area by using herbaceous plants | |
| CN111733100B (en) | Acinetobacter scherzei for degrading polyethylene mulching film and application thereof | |
| Sturini et al. | A preliminary investigation on Metal Bioaccumulation by Perenniporia fraxinea | |
| Fu et al. | Accumulation of copper in Trichoderma reesei transformants, constructed with the modified Agrobacterium tumefaciens-mediated transformation technique | |
| CN101250578A (en) | Method for culturing and screening microbiological bacterials | |
| CN103740593B (en) | A kind of anabena and application thereof of energy degradation of polychlorinated biphenyl | |
| CN103409346B (en) | A kind of heavy metal resistance Pectinatus and application thereof | |
| CN106282222A (en) | A kind of agriculture bacillus mediated pythium oligandrum genetic transforming method | |
| Mu et al. | Morphological and molecular identification of Pseudoplagiostoma castaneae sp. nov.(Pseudoplagiostomataceae, Diaporthales) in Shandong Province, China | |
| CN109423456B (en) | Azotobacter chroococcum as well as identification method and application thereof | |
| CN109423459B (en) | Pseudomonas and identification method and application thereof | |
| Alidadi et al. | Botryotrichum iranicum sp. nov. and Trematosphaeria magenta sp. nov. as two new species from Iran | |
| CN105483038A (en) | Aerobic arsenic methylation bacterium SM-1 of cytophagaceae and application of arsenic methylation bacterium | |
| CN115927077A (en) | Copper-greedy bacterium BERC-1 and application thereof | |
| Lababpour et al. | Isolation and submerged culture biomass production of the arid land cyanobacteria Microcoleus spp., an investigation on its utilization for biological soil crust restoration | |
| CN109439588A (en) | A kind of citric acid bacterial strain, pyridine biodegrade microbial inoculum and its preparation method and application | |
| CN103981121A (en) | Serratia marcescens M9 and application thereof to remove heavy metal ions | |
| CN109423458B (en) | Rhodococcus and identification method and application thereof | |
| Yu | Microbial Remediation of Heavy Metal (Ioid) Contaminated Soil: A Review | |
| CN109652337B (en) | It corrects high-flux sequence protokaryon and eukaryotic microorganisms gene order reads the method and bacteria used thereby of number | |
| CN102776128B (en) | Microbial compound bacteria agent for treating oilfield fracturing flowback fluid, and preparation method of microbial compound agent | |
| CN109423455B (en) | Rhodococcus equi, and identification method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20161005 |