CN108467842A - Bacterial strain and its application - Google Patents
Bacterial strain and its application Download PDFInfo
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- CN108467842A CN108467842A CN201810299046.6A CN201810299046A CN108467842A CN 108467842 A CN108467842 A CN 108467842A CN 201810299046 A CN201810299046 A CN 201810299046A CN 108467842 A CN108467842 A CN 108467842A
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- chlorate
- bacterial strain
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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
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/12—Halogens or halogen-containing compounds
Abstract
The present invention relates to microorganisms technical fields, more particularly to bacterial strain and its application.The bacterial strain is used to handle the pollutant chlorate in water body, and after the microbiological treatment, the pollutant chlorate in water body is fully converted to chlorion, has stronger biological prosthetic ability.Bacterial strain tolerable pH range when carrying out chloric acid salt respiration is wide, tolerable pH ranging from 4.5~8.0, specific growth rate is held in higher level under conditions of pH is 4.5~8.0, and growth is rapid, under conditions of pH is 5.0~8.0, which can 100% reduction chlorate.The bacterial strain carries out being resistant to NaClO when chloric acid salt respiration3Concentration is high, and highest can tolerate NaClO3A concentration of 132mM；In 120h, 100%, 100%, 88%, 47%, 48% degradation rate can be realized respectively to the chlorate that initial concentration is 9mM, 19mM, 24mM, 48mM.
Description
Technical field
The present invention relates to microorganisms technical fields, more particularly to bacterial strain and its application.
Background technology
Chlorate anions anion (ClO3-), or be a kind of virose strong oxidizer of tool, Ke Yiwen referred to as " chlorate "
Surely be present in water environment, have the characteristics that highly soluble, migration, and be not easy by soil, sedimentary particle absorption or
Biological aggregation.Chlorate by industrial production for many years, agriculturally, it is by as herbicide, defoliant, soil fungicides
And largely use, and applied as off-season production agent in anti-season YEAST IN LONGAN PRODUCTION；In the industry, it is high to be used to manufacture for it
Chlorate, chlorite are used as the oxidant and mordant of aniline printing, the bleaching agent being used as in chlorine dioxide production；It
It is the by-product using chlorine dioxide to water body disinfection.
The discharge standard not determined for chlorate at present, but before basis the study found that chlorate to some
Organism has apparent toxic effect.It has strong toxicity to many microorganisms and algae, and in drinking water higher dosage chloric acid
Salt may be one of inducement of anemia.
Mainly there are physical-chemical process and bioanalysis, wherein object for the minimizing technology of this environmental contaminants of chlorate at present
Change method includes absorption, ion exchange, membrane technology and electronation etc..General physico-chemical process cost is larger, and needs after-treatment.It is micro-
Low energy consumption for biological removal chlorate, efficient, does not need after-treatment, therefore have good feasibility, receives more next
More concerns.Biological prosthetic method, wherein the critically important part studied can exactly carry out chlorate
The microorganism of reduction.It is mainly used to restore the bacterium of chlorate at present to include chlorate reducing bacteria and perchlorate reduction bacterium,
Middle major part belongs to α, β, γ, ε in Proteobacteria deformation Gammaproteobacteria.The type of chlorate reducing bacteria is to influence chlorate reduction
It an important factor for effect, therefore the very necessary research for reinforcing screening chlorate reducing bacteria and cultivating, obtains different efficient
Dominant strain, the problem of to realize processing extensive such chlorate polluted-water.Although currently, having filtered out perhaps
The bacterium of chlorate can be mostly restored, such as：Dechlorospirillum、Magnetospirillum、Dechloromonas、
Dechlorobacter、Propionivibrio、Azospira、Dechlorosoma、Pseudomonas、
The bacterium that Dechloromarinus belongs to.But when the bacterium reported carries out chloric acid salt respiration at present not to the tolerance range of pH
It is enough wide in range, and the bacterium of most of pure cultures is in confused situation to the tolerance of higher concentration chlorate really.
Invention content
In view of this, a kind of bacterial strain of present invention offer and its application.The present invention carries out chlorate for existing bacterial strain at present
It is not wide in range enough to the tolerance range of pH when breathing, to the tolerance true deficiency in confused situation of higher concentration chlorate, provide one kind
It can be resistant to more wide pH value, higher concentration chlorate, and the new strains of chlorate can be restored, provide and a kind of disappeared using the bacterial strain
Except the method for environmental contaminants chlorate.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides bacterial strains, with any one in nucleotide sequence as follows：
I, there is SEQ ID NO:Nucleotide sequence shown in 1；
II, there is SEQ ID NO:Nucleotide sequence shown in 1 is through modifying, replacing, missing or adding one or more alkali
The nucleotide sequence that base obtains；
III and SEQ ID NO:Nucleotide sequence shown in 1 has the sequence of at least 80% homology；
The complementary series of IV, the sequence as shown in I, II or III.
In some specific embodiments of the present invention, bacterial strain deposit number provided by the invention is CCTCC NO：M
2018048。
The present invention also provides application of the bacterial strain in restoring chlorate.
The present invention also provides application of the bacterial strain in administering sewage.
In some specific embodiments of the present invention, the sewage contains chlorate.
The present invention also provides a kind of reduction chlorate and/or the methods for handling the sewage containing chlorate, will be of the invention
The bacterial strain activation of offer is inoculated in after expanding culture in the culture medium or sewage containing chlorate.
In some specific embodiments of the present invention, the bacterial strain is 4.5 to the minimum tolerance value of pH.
In some specific embodiments of the present invention, the pH of the culture medium or sewage is 4.5~8.0.
In some specific embodiments of the present invention, the bacterial strain is 132mM to the tolerable concentration of chlorate.
In some specific embodiments of the present invention, the method is specially：By bacterial strain single bacterium colony provided by the invention
It is inoculated in 3mL LB and cultivates 12h, then be transferred in 100mL LB and cultivate 12h, collect bacterium and be added to and electronics is made with sodium chlorate
In the minimal medium of receptor, 240h is cultivated, timing sampling measures bacterial growth situation in solution, perchlorate concentration changes,
Chlorine ion concentration changes.
In some specific embodiments of the present invention, LB medium components are (g/L)：Tryptone 10；Yeast extracts
Object 5；NaCl10.
In some specific embodiments of the present invention, minimal medium is (g/L)：NH4Cl0.25；NaH2PO4·
2H2O 0.78；KCl0.1；NaHCO32.5；NaClO31.064；Glucose 1.802.
In some specific embodiments of the present invention, the initial OD for bacterial strain described in minimal medium of transferring600About
It is 0.1.
In some specific embodiments of the present invention, spectrophotometric determination OD is used600Growth curve of bacteria is drawn,
The variation of Chlorine in Solution hydrochlorate concentration is measured using ion chromatography instrument, chlorine ion concentration changes.The bacterial strain is in pH4.5~8.0
Under conditions of, electron acceptor is made with sodium chlorate, may be grown.Under conditions of 4.5~8.0 pH, the ratio growth speed of bacterium
Rate is held in higher level, and growth is rapid.Except under conditions of pH is 4.5, which only restores 74% chlorate, is in pH
Under conditions of 5.0~8.0, which can 100% reduction chlorate.
In some specific embodiments of the present invention, the method is specially：By bacterial strain single bacterium colony provided by the invention
It is inoculated in 3mL LB and cultivates 12h, then be transferred in 100mL LB and cultivate 12h, collect bacterium and be added to and electronics is made with sodium chlorate
In the minimal medium of the different pH of receptor, timing sampling measures the growth curve and chlorate also virgin curve of bacterium.
In some specific embodiments of the present invention, buffer system is the phosphoric acid of 100mM in the minimal medium
Salt.
In some specific embodiments of the present invention, the bacterial strain highest can tolerate NaClO3A concentration of 132mM.
In 120h, can realize 100% respectively to the chlorate that initial concentration is 9mM, 19mM, 24mM, 48mM, 100%, 88%, 47%,
48% degradation rate.
In some specific embodiments of the present invention, the method is specially：Isolated bacterial strain single bacterium colony is connect
Kind cultivates 12h in 3mL LB, then is transferred in 100mL LB and cultivates 12h, collects bacterium and is added to various concentration sodium chlorate
In minimal medium, timing sampling measures bacterial growth situation in solution.
The preparation of the sewage containing chlorate the present invention also provides a kind of reduction chlorate and/or processing, including this hair
The bacterial strain of bright offer and acceptable auxiliary material.
The present invention screens the human pallid bacillus strain X M-1 for obtaining to restore chlorate from micropopulation, which behaves
First plant of bacterium for being found to have reduction chlorate ability in Ochrobactrum.The bacterium can go back completely under suitable conditions
Pollutant chlorate is chlorion in raw water body.
A kind of microbial strains disclosed by the invention, the microbial strains are XM-1, and Classification And Nomenclature is human pallid bacillus
(Ochrobacterum anthropi) is CCTCC NO in the preserving number of China typical culture collection center：M2018048.
Tolerable pH range is wide when the human pallid bacillus strain X M-1 progress chloric acid salt respirations that the present invention screens, and can tolerate
PH ranging from 4.5~8.0.Compared to the chlorate reducing bacteria filtered out in the past, which is resistant to the ability of acid condition obviously more
By force, and under conditions of 4.5~8.0 pH, the specific growth rate of bacterium is held in higher level, and growth is rapid, and divides before
Under conditions of pH is less than 7, the specific growth rate of bacterium significantly reduces the chlorate reducing bacteria separated out；Except the item for being 4.5 in pH
Under part, the bacterium only degrade 74% chlorate, pH be 5.0~8.0 under conditions of, the bacterium can 100% degrade chlorate.Cause
This bacterium is in the chlorate of practical water body of the reduction with wide pH value with application prospect, especially acid range water body.
The human pallid bacillus strain X M-1 that the present invention screens carries out being resistant to NaClO when chloric acid salt respiration3Concentration is high, most
The tolerable NaClO of height3A concentration of 132mM can realize 132mM chlorates about 48% degradation in 120h.The bacterium is restoring
There is application prospect in high concentration cl hydrochlorate water.
Biological deposits explanation
Biomaterial：Human pallid bacillus XM-1；Classification And Nomenclature：Human pallid bacillus XM-1 (Ochrobacterum
anthropi XM-1)；It was preserved in China typical culture collection center on 01 22nd, 2018, collection address is：In
Wuhan Wuhan University of state；Deposit number is CCTCC NO：M2018048.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described.
Fig. 1 shows the phylogenetic tree of bacterial strain of the present invention；
Fig. 2 shows bacterial strain of the present invention degradation curve of chlorate, chlorion formation curve, thin in minimal medium
Bacterium growth curve in inorganic salts；
Fig. 3 shows growth curve of the bacterial strain of the present invention under condition of different pH；
Fig. 4 shows maximum specific growth rate of the bacterial strain of the present invention under condition of different pH；
Fig. 5 shows the degradation curve of chlorate of the bacterial strain of the present invention under condition of different pH；
Fig. 6 shows growth curve of the bacterial strain of the present invention in various concentration chlorate；
Fig. 7 shows the degradation curve of chlorate of the bacterial strain of the present invention in various concentration chlorate.
Specific implementation mode
The invention discloses a kind of bacterial strain and its application, those skilled in the art can use for reference present disclosure, be suitably modified
Technological parameter is realized.In particular, it should be pointed out that all similar substitutions and modifications be for a person skilled in the art it is aobvious and
It is clear to, they are considered as being included in the present invention.The method of the present invention and application are retouched by preferred embodiment
It states, related personnel can obviously not depart from the content of present invention, change to method described herein and application in spirit and scope
It moves or suitably changes and combine, to realize and apply the technology of the present invention.
It is not wide in range enough to the tolerance range of pH when for current existing bacterial strain progress chloric acid salt respiration, to higher concentration chloric acid
The tolerance true deficiency in confused situation of salt, more wide pH value, higher concentration chlorate can be resistant to by providing one kind, and can restore chlorine
The new strains of hydrochlorate provide a kind of method for eliminating environmental contaminants chlorate using the bacterial strain.
The initial inoculation object that the present invention uses derives from the oxidation ditch process of Hefei City, Anhui Province Wang Tang sewage treatment plants
Activated sludge, interior use in one week after sampling.
The present invention is enriched with required bacterial strain using liquid selective medium concentration method.Take 5g sludge seedings to anaerobism serum bottle
In, 50mL minimal mediums are added into serum bottle, with NaClO3(10mM) makees electron acceptor, CH3COONa (10mM) makees electricity
Sub- donor.Minimal medium ingredient is (g/L)：NH4Cl 0.25；NaH2PO4·2H2O 0.78；KCl0.1；NaHCO32.5；
10mL trace elements, 10mL vitamins.Trace element suite becomes (g/L)：Nitrilotriacetic acid 1.5；MgSO43.0；MnSO4·H2O
0.5；NaCl 1.0；FeSO4·7H2O 0.1；CaCl2·2H2O 0.1；CoCl2·6H2O 0.1；ZnCl 0.13；CuSO4
0.01；AlK(SO4)2·12H2O 0.01；H3BO20.01；Na2MoO40.025；NiCl2·6H2O 0.024；Na2WO4·
2H2O 0.025.Vitamin group becomes (mg/L)：Biotin 2；Folic acid 2；Vitamin B2 5；Vitamin B1 5；Niacin 5；Dimension life
Plain B5 5；Vitamin B12 0.1；P-aminobenzoic acid 5.Medium pH is about 7.2, be not necessarily to additional tune pH the step of.Use N2/
CO2Gas (N2:CO2=8：2) 20min is aerated to culture medium in serum bottle, anaerobism ring in bottle is kept to remove the oxygen in bottle
Then border sterilizes (121 DEG C, 20min).In serum bottle after primary vaccination sludge, it is put into culture in shaking table (30 DEG C, 200rpm)
10d；Second of switching takes 10% of mixture in the last serum bottle transferred, is transferred in fresh culture medium, is put into shaking table
Culture in (30 DEG C, 200rpm)；It is continuously transferred, after switching is more than or equal to three times, is passed through in the same way later
It is incubated overnight, solution appeared cloudy state in serum bottle, stops enriching step.
The present invention detaches required bacterial strain using solid selection medium.Take the serum transferred for the last time in enriching step
Culture medium in bottle, is coated on inorganic salts solid medium, inorganic salts solid medium is with NaClO3(10mM) make electronics by
Body, CH3COONa (10mM) makees electron donor, and 1.5%Agar is added.By the single bacterium colony to come in every shape grown on tablet point
Do not choose, then obtains purebred bacterium by the step of constantly purifying.
By verifying the ability of isolated purebred bacterial reduction chlorate in the present invention, aimed strain is obtained.Select institute
Bacterium single bacterium colony, in the LB culture mediums for entering 3mL, LB medium components are (g/L)：Tryptone 10；Yeast extract 5；
NaCl 10 is put into shaking table (30 DEG C, 200rpm) and cultivates 12h.Take out quantitative (1mL, OD600It is transferred to for bacterium 2.472)
In the LB culture mediums of 100mL, it is put into shaking table (30 DEG C, 200rpm) and cultivates 12h.By the bacterium in the LB of cultured 100mL
It centrifuges (6000g, 5min), removes supernatant collection thalline；It washed once using minimal medium, centrifugation (6000g,
5min), supernatant collection thalline is removed；Bacterium is resuspended using minimal medium, is injected into and is trained containing 50mL inorganic salts
(control bacterium initial OD is supported in the serum bottle of base600About 0.1), NaClO3(10mM) makees electron acceptor, and glucose (10mM) is made
Electron donor is put into culture, timing sampling in shaking table (30 DEG C, 200rpm).Aimed strain can be grown in above-mentioned culture medium,
And perchlorate concentration reduces at any time in culture medium, judgement obtains aimed strain.
By measuring the 16S rDNA sequences of isolated strains, division bacteria is determined.Bacterial strain DNA is extracted, primer 2 7F is utilized
The 16S rDNA genes that the bacterial strain is expanded with 1492R, product is connect with carrier T, confirms the fragment sequence through sequencing.By the sequence
Row are compared with related data in GenBank, and the bacterium is homologous with human pallid bacillus (Ochrobacterum anthropi)
Property reaches 99%.It can confirm that the bacterial strain is human pallid bacillus (Ochrobacterum anthropi), be named as XM-1.
In China typical culture collection center, deposit number is CCTCC NO for the microbial preservation：M 2018048.
The present invention measures growing state of the gained bacterium under condition of different pH and also by changing minimal medium pH
Former chlorate situation.With NaClO in minimal medium3(10mM) makees electron acceptor, and glucose (10mM) makees electron donor, will
The buffer salt of bicarbonate-dihydric phosphate changes the phosphate buffer salt of 100mM into former culture medium, adjusts culture medium respectively
PH is 4.0,4.5,5.0,6.0,6.5,7.0,7.5,8.0,9.0.The result shows that the bacterium can tolerate pH ranging from 4.5~8.0,
Under conditions of pH4.5~8.0, the specific growth rate of bacterium is held in higher level, and growth is rapid；Except being 4.5 in pH
Under the conditions of, which only restores 74% chlorate, and under conditions of pH is 5.0~8.0, which can 100% reduction chlorate.
The present invention is by changing electron donor NaClO3Concentration, measure gained bacterium in various concentration NaClO3Condition
Under growing state and reduction chlorate situation.The result shows that the bacterium highest can tolerate NaClO3A concentration of 132mM；In 120h
It is interior, 100%, 100%, 88%, 47%, 48% can be realized respectively to the chlorate that initial concentration is 9mM, 19mM, 24mM, 48mM
Degradation rate.
The beneficial effects of the invention are as follows：
One, present invention screening from micropopulation obtains to restore the human pallid bacillus strain X M-1 of chlorate, the bacterium
The bacterium for being found to have reduction chlorate ability for first plant in people's Ochrobactrum.The bacterium under suitable conditions can be complete
Pollutant chlorate is chlorion in full reduction water body.
Tolerable pH range is wide when the human pallid bacillus strain X M-1 progress chloric acid salt respirations that two, present invention screening obtains, can
Tolerable pH range is 4.5~8.0.Compared to the chlorate reducing bacteria filtered out in the past, the ability which is resistant to acid condition is bright
It is aobvious stronger, and under conditions of pH4.5~8.0, the specific growth rate of bacterium is held in higher level, and growth is rapid, and it
Before the chlorate reducing bacteria isolated under conditions of pH is less than 7, the specific growth rate of bacterium significantly reduces；Except pH be 4.5
Under conditions of, the bacterium only degrade 74% chlorate, pH be 5.0~8.0 under conditions of, the bacterium can 100% degrade chloric acid
Salt.Therefore the bacterium reduction the practical water body with wide pH value chlorate in application prospect, especially acid range water
Body.
Three, the human pallid bacillus strain X M-1 that present invention screening obtains carries out being resistant to NaClO when chloric acid salt respiration3Concentration
Height, highest can tolerate NaClO3A concentration of 132mM can realize 132mM chlorates about 48% degradation in 120h.The bacterium
There is application prospect in reduction high concentrate chloric acid brine.
Raw material and reagent are available on the market used in the process of the entire experiment of bacterial strain provided by the invention and its application.
With reference to embodiment, the present invention is further explained：
Embodiment 1：The screening of microbial strains of the present invention
Liquid selective medium concentration method：Initial inoculation object uses the oxidation ditch of Hefei City, Anhui Province Wang Tang sewage treatment plants
The activated sludge of technique takes in 5g sludge seedings to anaerobism serum bottle, and 50mL minimal mediums are added into serum bottle, with
NaClO3(10mM) makees electron acceptor, CH3COONa (10mM) makees electron donor.Medium pH is about 7.2, is not necessarily to additional tune pH
The step of.Use N2/CO2Gas (N2:CO2=8：2) 20min is aerated to culture medium in serum bottle, to remove the holding of the oxygen in bottle
Then anaerobic environment in bottle sterilizes (121 DEG C, 20min).In serum bottle after primary vaccination sludge, be put into shaking table (30 DEG C,
10d is cultivated in 200rpm)；Second of switching takes 10% of mixture in the last serum bottle transferred, is transferred to fresh culture
In base, it is put into culture in shaking table (30 DEG C, 200rpm)；Continuously transferred in the same way later, when switching be more than etc.
After three times, by being incubated overnight, solution appeared cloudy state in serum bottle, explanation may have aimed strain to be enriched with.
Solid selection medium partition method：The culture medium in the serum bottle transferred for the last time in enriching step is taken, is coated with
In on inorganic salts solid medium, inorganic salts solid medium is with NaClO3(10mM) makees electron acceptor, CH3COONa (10mM) makees
1.5%Agar is added in electron donor.The single bacterium colony to come in every shape grown on tablet is chosen respectively, is then passed through continuous
Ground is crossed on solid medium, purifying gained bacterium.By the above method, the present invention filters out one plant of microbial strains XM-
1。
The minimal medium ingredient is (g/L)：NH4Cl 0.25；NaH2PO4·2H2O 0.78；KCl 0.1；
NaHCO32.5；10mL trace elements, 10mL vitamins.Trace element suite becomes (g/L)：Nitrilotriacetic acid 1.5；MgSO43.0；
MnSO4·H2O 0.5；NaCl 1.0；FeSO4·7H2O 0.1；CaCl2·2H2O 0.1；C℃l2·6H2O 0.1；ZnCl
0.13；CuSO40.01；AlK(SO4)2·12H2O 0.01；H3BO20.01；Na2MoO40.025；NiCl2·6H2O
0.024；Na2WO4·2H2O 0.025.Vitamin group becomes (mg/L)：Biotin 2；Folic acid 2；Vitamin B2 5；Vitamin B1
5；Niacin 5；Vitamin B5 5；Vitamin B12 0.1；P-aminobenzoic acid 5.
Embodiment 2：The identification of microbial strains of the present invention
Bacterial strain DNA is extracted according to raw work Ezup pillar bacterial genomes DNA extraction agent box steps, utilizes primer：
27F：5 '-AGAGTTTGATCCTGGCTCAG-3 ' and 1492R：5 '-GGTTACCTTGTTACGACTT-3 ' expand the bacterial strain
Product is connect by 16S rDNA genes with carrier T, confirms that the segment physical length is 1443bp through sequencing.By the sequence with
Related data is compared in GenBank, in comparing the highest result of matching degree, the 16S rDNA sequences that are arrived measured by the bacterial strain
Row part and the homology of human pallid bacillus (Ochrobacterum anthropi) reach 99%, and it is grey can to confirm that the bacterial strain is behaved
White bacillus (Ochrobacterum anthropi), is named as XM-1.
According in the 16S rDNA gene orders (as shown in SEQ ID No.1) and GenBank of the isolated strains measured
With the 16S of other higher bacteriums of its comparison result matching degree and other (height) chlorate reducing bacterias more reported in the literature
RDNA gene orders make the phylogenetic tree of isolated strains according to adjacent method, as shown in Figure 1 using MEGA6 softwares.By Fig. 1
It is found that the overwhelming majority (height) the chlorate reducing bacteria being currently known belongs to Proteobacteria, it is mainly distributed on β deformation Gammaproteobacterias and α becomes
Shape Gammaproteobacteria, this plant of bacterium that the present invention isolates belongs to the Ochrobactrum below α deformation Gammaproteobacterias, currently without Ochrobactrum
In other bacteriums be reported have reduction chlorate ability.
Embodiment 3：The method for restoring chlorate using strain X M-1
It selects in the LB culture mediums that the bacterial strain monoclonal enters 3mL, 12h is cultivated in shaking table (30 DEG C, 200rpm).It is fixed to take out
Measure (1mL, OD600It is transferred in the LB culture mediums of 100mL for bacterium 2.472), is put into culture in shaking table (30 DEG C, 200rpm)
12h.Bacterium in the LB of cultured 100mL is centrifuged into (6000g, 5min), removes supernatant collection thalline；Use inorganic salts
Culture medium washed once, and centrifuge (6000g, 5min), remove supernatant collection thalline；Bacterium is resuspended using minimal medium,
(control bacterium initial OD is injected into the serum bottle containing 50mL minimal mediums600About 0.1), NaClO3Make electricity
Sub- receptor, glucose make electron donor, are put into shaking table (30 DEG C, 200rpm) and co-culture 240h, and timing sampling.
Use spectrophotometric determination sample OD600Describe growth curve, is measured in culture medium using ion chromatography instrument
Perchlorate concentration, as a result as shown in Fig. 2, table 1.In the 120h of culture, which can utilize glucose and NaClO3It carries out
Respiration realizes the growth of bacterium, which can be completely converted into chlorion by sodium chlorate all in culture medium.
Table 1
Embodiment 4：The case where growing states of the strain X M-1 under condition of different pH and reduction chlorate
Isolated pure culture bacterial strain is selected in the LB culture mediums that monoclonal enters 3mL, in shaking table (30 DEG C, 200rpm)
Middle culture 12h.It takes out quantitative bacterium to be transferred in the LB culture mediums of 100mL, is put into shaking table (30 DEG C, 200rpm) and cultivates 12h.
Bacterium in the LB of cultured 100mL is centrifuged into (6000g, 5min), removes supernatant collection thalline；Use inorganic salts culture
Base washed once, and centrifuge (6000g, 5min), remove supernatant collection thalline；Bacterium is resuspended using minimal medium, by it
It is injected separately into (control in the minimal medium for being 4.0,4.5,5.0,6.0,6.5,7.0,7.5,8.0,9.0 containing 50mL pH
Bacterium initial OD processed600About 0.1).Minimal medium is with NaClO3(10mM) makees electron acceptor, and glucose (10mM) makees electricity
The buffer salt of bicarbonate-dihydric phosphate in former culture medium is changed into the phosphate buffer salt of 100mM, adjusted respectively by sub- donor
It is 4.0,4.5,5.0,6.0,6.5,7.0,7.5,8.0,9.0 to save pH.
Timing sampling measures the growth curve and chlorate also virgin curve of bacterium.As shown in Fig. 3, table 2~3, which can be with
It is grown under conditions of pH4.5~8.0.As shown in Fig. 4, table 4, under conditions of pH4.5~8.0, the ratio of bacterium is grown
Rate is held in higher level, and growth is rapid.As shown in Fig. 5, table 5~6, except under conditions of pH is 4.5, which only restores
74% chlorate, under conditions of pH is 5.0~8.0, which can 100% reduction chlorate.
Table 2
Table 3
Table 4
Table 5
Table 6
Embodiment 5：The case where growing states of the strain X M-1 in various concentration chloric acid salt environment and reduction chlorate
Isolated pure culture bacterial strain is selected in the LB culture mediums that monoclonal enters 3mL, in shaking table (30 DEG C, 200rpm)
Middle culture 12h.It takes out quantitative bacterium to be transferred in the LB culture mediums of 100mL, is put into shaking table (30 DEG C, 200rpm) and cultivates 12h.
Bacterium in the LB of cultured 100mL is centrifuged into (6000g, 5min), removes supernatant collection thalline；Use inorganic salts culture
Base washed once, and centrifuge (6000g, 5min), remove supernatant collection thalline；Bacterium is resuspended using minimal medium, by it
(control bacterium initial OD is injected separately into the minimal medium of the different perchlorate concentrations containing 50mL600About 0.1).
Minimal medium makees electron donor with glucose (10mM).
Timing sampling measures the growth curve and chlorate also virgin curve of bacterium.As shown in Fig. 6, table 7, which can
It is resistant to NaClO3A concentration of 132mM.Can be 9mM, 19mM, 24mM, 48mM to initial concentration in 120h as shown in Fig. 7, table 8
Chlorate realize respectively 100%, 100%, 88%, 47%, 48% degradation rate.
Table 7
Table 8
Comparative example
Condition of culture is the same as embodiment 4.
The maximum growth rate of bacterium is as shown in table 9 under different pH condition：
Table 9
Under the conditions of same pH, the bacterial strain of comparative example and the maximum growth rate of bacterial strain provided by the invention have extremely significantly
Difference (P ＜ 0.01), shows that bacterial strain provided by the invention is significantly better than the tolerance pole of pH value the bacterial strain of comparative example.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Sequence table
<110>China Science ＆ Technology University
<120>Bacterial strain and its application
<130> MP1800943
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1443
<212> DNA
<213>Human pallid bacillus (Ochrobactrum anthropic)
<400> 1
gagtttgatc ctggctcaga acgaacgctg gcggcaggct taacacatgc aagtcgagcg 60
ccccgcaagg ggagcggcag acgggtgagt aacgcgtggg aacgtacctt ttgctacgga 120
ataactcagg gaaacttgtg ctaataccgt atgtgccctt cgggggaaag atttatcggc 180
aaaggatcgg cccgcgttgg attagctagt tggtgaggta aaggctcacc aaggcgacga 240
tccatagctg gtctgagagg atgatcagcc acactgggac tgagacacgg cccagactcc 300
tacgggaggc agcagtgggg aatattggac aatgggcgca agcctgatcc agccatgccg 360
cgtgagtgat gaaggcccta gggttgtaaa gctctttcac cggtgaagat aatgacggta 420
accggagaag aagccccggc taacttcgtg ccagcagccg cggtaatacg aagggggcta 480
gcgttgttcg gatttactgg gcgtaaagcg cacgtaggcg gacttttaag tcaggggtga 540
aatcccgggg ctcaaccccg gaactgcctt tgatactgga agtcttgagt atggtagagg 600
tgagtggaat tccgagtgta gaggtgaaat tcgtagatat tcggaggaac accagtggcg 660
aaggcggctc actggaccat tactgacgct gaggtgcgaa agcgtgggga gcaaacagga 720
ttagataccc tggtagtcca cgccgtaaac gatgaatgtt agccgttggg gagtttactc 780
ttcggtggcg cagctaacgc attaaacatt ccgcctgggg agtacggtcg caagattaaa 840
actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca 900
acgcgcagaa ccttaccagc ccttgacata ccggtcgcgg acacagagat gtgtctttca 960
gttcggctgg accggataca ggtgctgcat ggctgtcgtc agctcgtgtc gtgagatgtt 1020
gggttaagtc ccgcaacgag cgcaaccctc gcccttagtt gccagcattt agttgggcac 1080
tctaagggga ctgccggtga taagccgaga ggaaggtggg gatgacgtca agtcctcatg 1140
gcccttacgg gctgggctac acacgtgcta caatggtggt gacagtgggc agcgagcacg 1200
cgagtgtgag ctaatctcca aaagccatct cagttcggat tgcactctgc aactcgagtg 1260
catgaagttg gaatcgctag taatcgcgga tcagcatgcc gcggtgaata cgttcccggg 1320
ccttgtacac accgcccgtc acaccatggg agttggtttt acccgaaggc gctgtgctaa 1380
ccgcaaggag gcaggcgacc acggtagggt cagcgactgg ggtgaagtcg taacaaggta 1440
acc 1443
Claims (10)
1. bacterial strain, which is characterized in that with any one in nucleotide sequence as follows：
I, there is SEQ ID NO:Nucleotide sequence shown in 1；
II, there is SEQ ID NO:Nucleotide sequence shown in 1 is obtained through modifying, replacing, missing or adding one or more bases
The nucleotide sequence obtained；
III and SEQ ID NO:Nucleotide sequence shown in 1 has the sequence of at least 80% homology；
The complementary series of IV, the sequence as shown in I, II or III.
2. bacterial strain according to claim 1, which is characterized in that its deposit number is CCTCC NO：M 2018048.
3. application of the bacterial strain according to claim 1 or 2 in restoring chlorate.
4. application of the bacterial strain according to claim 1 or 2 in administering sewage.
5. application according to claim 4, which is characterized in that the sewage contains chlorate.
6. a kind of reduction chlorate and/or the method for handling the sewage containing chlorate, which is characterized in that will be such as claim 1
Or bacterial strain activation, the expansion described in 2 are inoculated in after cultivating in the culture medium or sewage containing chlorate.
7. according to the method described in claim 6, it is characterized in that, the bacterial strain is 4.5 to the minimum tolerance value of pH.
8. the method described according to claim 6 or 7, which is characterized in that the pH of the culture medium or sewage is 4.5~8.0.
9. according to claim 6 to 8 any one of them method, which is characterized in that tolerable concentration of the bacterial strain to chlorate
For 132mM.
10. a kind of reduction chlorate and/or the preparation for handling the sewage containing chlorate, which is characterized in that wanted including such as right
Ask the bacterial strain described in 1 or 2 and acceptable auxiliary material.
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| CN110724650A (en) * | 2019-10-21 | 2020-01-24 | 天津大学 | Efficient petroleum degrading bacterium TDYN1T and application thereof |
| WO2021077531A1 (en) * | 2019-10-21 | 2021-04-29 | 天津大学 | Efficient petroleum degradation bacteria tdyn1t and use thereof |
| CN110724650B (en) * | 2019-10-21 | 2021-11-02 | 天津大学 | Petroleum degrading bacterium TDYN1T and application thereof |
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