CN107858309A - Triazole degraded bacillus and its application - Google Patents
Triazole degraded bacillus and its application Download PDFInfo
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- CN107858309A CN107858309A CN201711249249.6A CN201711249249A CN107858309A CN 107858309 A CN107858309 A CN 107858309A CN 201711249249 A CN201711249249 A CN 201711249249A CN 107858309 A CN107858309 A CN 107858309A
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- triazole
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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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- 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
- C12R2001/07—Bacillus
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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
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
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- Hydrology & Water Resources (AREA)
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
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Abstract
The invention discloses one plant of triazole degraded bacillus and its application in waste water containing triazole.The present invention uses the enrichment that triazole degradation bacteria is carried out using triazole as sole carbon source and the culture medium of nitrogen source, and the screening and culturing medium using triazole as sole carbon source and nitrogen source is used to be separated, obtain triazole degraded bacillus, Bacillus sp.NJUST43 are named as, deposit number is CCTCC NO：M 2017102.The Bacillus sp.NJUST43 bacterial strains of the present invention can realize the processing for the triazole waste water that concentration is about 150mg/L in 15d or so, and the degradation rate of triazole reaches more than 90%, have preferably tolerance performance to triazole, have a good application prospect.
Description
Technical field
The invention belongs to environmental organic pollutant biologic treating technique field, and in particular to one plant of triazole degraded gemma bar
Bacterium and its application in biological wastewater treatment and environmental pollution are repaired.
Background technology
Triazole has stronger water solubility because of its heterocycle structure, it is easy to is transferred in soil and groundwater, in soil
Generally existing in earth and underground water, it is a kind of typical difficult degradation nitrogen-containing heterocycle compound.Triazole has bio-toxicity, teratogenesis
Change and carcinogenic nature, into environment, will seriously endanger health.Therefore, after need to handling the waste water containing triazole
Discharge.
Mainly there are Physical, chemical method, bioanalysis etc. currently for the processing method of triazole waste water.Wherein physical means
Such as absorption method, extraction really effectively can not be removed triazole completely, it is necessary to be aided with other techniques, just can reach ideal
Clearance；The middle-and-high-ranking oxidizing process of chemical method and electrochemical process power consumption are high, and cost is also high, and the toxicity of partial intermediate is more
Greatly, secondary pollution is easily produced.Therefore, high treating effect, processing cost small, non-secondary pollution the waste water containing triazole are studied
Treatment technology and technique, there is important theoretical and practical significance.
Microorganism treatment has the advantages of economic, efficient, it is often more important that Ke Yishi compared with physics, chemical method
The innoxious governance now polluted.Bioanalysis non-secondary pollution, treating capacity are big, are wastewater processing technologies most widely used at present.But
Because triazole structure is stable, bio-toxicity is big, and bio-degradable degree is not high, and common biological treatment can not act on or efficiency
Lowly.Therefore, high to triazole degradation efficiency, good degrading effect microorganism is tamed and screened, is to solve the dirt of triazole waste water
The effective way of dye.
The content of the invention
It is an object of the invention to the shortage for current triazole degradation bacteria strains, there is provided one plant of triazole degraded gemma bar
Bacterium Bacillus sp.NJUST43.
Inventor's separation screening from the sludge of the biochemical treatment system of laboratory long-term degradation waste water containing triazole obtains
Triazole degradation bacteria strains NJUST43, is identified as Bacillus sp., is named as Bacillus sp.NJUST43, GenBank
The number of logging in is MF966511, and bacterial strain is on March 9th, 2017 in China typical culture collection center (CCTCC) preservation, preservation
Numbering is CCTCC NO：M 2017102.
The present invention also provides above-mentioned triazole and degrades bacillus sp.NJUST43 at waste water containing triazole
Application in reason.
Applications of the above-mentioned nitrogen azoles degraded bacillus sp.NJUST43 in wastewater treatment containing triazole,
Specific method is：Nitrogen azoles degraded bacillus sp.NJUST43 seed liquors are inoculated into waste water containing triazole, trained
It is 6.0-8.0 to support pH, and cultivation temperature is 25-35 DEG C.
Compared with prior art, the present invention has advantages below：
The Bacillus sp.NJUST43 of the present invention, can be grown by sole carbon source, nitrogen source of triazole.Screening
Obtained Bacillus sp.NJUST43, can realize the processing for the triazole waste water that concentration is about 150mg/L in 15d or so, three
The degradation rate of nitrogen azoles reaches more than 90%.In the range of triazole concentration is 0-150mg/L, Bacillus sp.NJUST43
Normal growth and the degradable of triazole can be realized.In the range of pH is 6.0-8.0, Bacillus sp.NJUST43 can
The degradable of triazole is realized, the addition of the degradable carbon source of low concentration can degrade to triazole to play a driving role.
Brief description of the drawings
Fig. 1 is influence of the triazole concentration to bacterial strain Bacillus sp.NJUST43 degraded triazole performances in waste water.
Fig. 2 is influence of the wastewater pH to triazole degradation property.
Fig. 3 is influence of the presence of degradable carbon source in waste water to triazole degradation property.
Embodiment
The following examples and accompanying drawing can make those skilled in the art that the present invention be more fully understood, but not with any side
The formula limitation present invention.
Embodiment 1：Triazole degradation bacteria Bacillus sp.NJUST43 screening separation and its degradability to triazole
Energy
(1) the separation of bacterial strain
Sludge aggregate sample is taken in the sludge of the biochemical treatment system of laboratory long-term degradation waste water containing triazole, takes 50mL dirty
Mud aggregate sample adds the liquid inorganic salt culture medium (MSM) that 100mL triazoles concentration is 100mg/L, loads 250mL triangular flasks
In, take out triangular flask with 180 revs/min of rotating speed shaking table culture under the conditions of 30 DEG C, after 3h and stand, taken after mud-water separation on 2mL
Clear liquid, 50mL triazoles concentration of transferring are in 100mg/L liquid inorganic salt culture medium (MSM), load 100mL triangular flasks
In, with 180 revs/min of rotating speed shaking table culture under the conditions of 30 DEG C, realize the enrichment culture of triazole degradation bacteria.After 15 days, 2mL liquid
Body culture medium is transferred into 50mL fresh cultures, and shaking table culture.After continuous 3 times switchings, with sterile distilled water by liquid
Culture medium dilution 104-108Times, it is 100mg/L, using triazole as sole carbon source and the inorganic salts of nitrogen source to be coated on triazole concentration
On solid medium flat board, it is put into 30 DEG C of incubators and is cultivated.Picking has the bacterium of notable difference on colony characteristicses after one week
Fall, using plate streaking separate method purified, continuous purification three times after, obtain single bacterial strain, and carry out inclined-plane preservation.
The composition of minimal medium (MSM) is as follows：Na2HPO4·12H2O (3.057g/L), KH2PO4(0.760g/L),
MgSO4·7H2O (0.2g/L), CaCl2(0.05g/L), trace element solution SL-4 (10mL/L).Micro- SL-4 compositions：
EDTA(0.5g/L),FeSO4·7H2O (0.2g/L), micro- SL-6 (100mL/L).Micro- SL-6 compositions：
ZnSO4·7H2O (0.01g/L), MnCl2·4H2O (0.03g/L), H3BO4(0.3g/L), CoCl2·6H2O (0.2g/L),
CuCl2·2H2O (0.01g/L), NiCl2·6H2O (0.02g/L), Na2MoO4·2H2O(0.03g/L)。
(2) the screening of bacterial strain
The single bacterium colony of picking resulting separation, it is 100mg/L, using triazole as sole carbon to be inoculated in triazole concentration respectively
In the MSM of source and nitrogen source, shaking table culture 15d.With triazole change in concentration in high effective liquid chromatography for measuring culture medium, if training
Supporting triazole concentration in base significantly reduces, you can shows that triazole is degraded.In isolated single bacterium colony, it is named as
NJUST43 strains for degrading performance is the most excellent, and in 15d culture period, triazole is degradable, therefore chooses bacterial strain work
Follow-up identification.
(3) the identification of bacterial strain
Morphology, Physiology and biochemistry test are carried out to bacterium.The 16S rDNA sequences of bacterial strain are determined, by the 16S rDNA of bacterial strain
Gene order carries out online tetraploid rice with the sequence in international GenBank databases, and finally being determined from molecular level should
The kind of bacterium.
1. morphological feature：NJUST43 bacterium colonies are white, and circular, there is fold at neat in edge, center.
2. physiological and biochemical property：Gram-positive, catalyzing enzyme is negative, oxidase positive, contacts enzyme positive, it is impossible to which hydrolysis is formed sediment
Powder, most suitable degraded pH scopes are 6.5-7.5, and optimum growth temperature is 25-35 DEG C.
3. molecular biology identification：Using the core DNA of NJUST43 bacterium as template, expanded with the PCR of 16S rDNA genes logical
It is primer with primer, enters performing PCR amplification, determine its complete sequence, the 16S rDNA gene orders of the bacterial strain are shown in sequence table.By bacterial strain
16S rDNA gene orders be committed to GenBank databases (the GenBank numbers of logging in are MF966511), with GenBank data
Sequence in storehouse carries out online tetraploid rice, the results showed that, NJUST43 and Bacillus cereus strain
HlS1200907 and Bacillus sp.strain FJAT-25493 sequence similarity is up to more than 99%.
Gemma is accredited as according to NJUST43 morphology, Physiology and biochemistry test and molecular biological analysis, NJUST43
Bacillus Bacillus sp., are named as Bacillus sp.NJUST43.
(4) degraded of the bacterial strain to triazole and the application in triazole wastewater treatment
Triazole degradation bacteria strains NJUST43 is seeded to the MSM culture mediums of addition 100mg/L triazoles, under the conditions of 30 DEG C
With 180 revs/min of rotating speed shaking table culture, NJUST43 enrichment culture is carried out, treats that thalline enters the exponential phase later stage (about
7d), by gained thalline with 3,000 × g rotating speed centrifuges 5 minutes, skims supernatant, using the method for the concussion that is vortexed by bacterium
Body is resuspended in sterile liquid MSM, centrifugation.After repeated washing process four times, thalline is resuspended in sterile liquid MSM
(the MSM amounts that regulation adds, control bacterium suspension optical density OD600About 0.2), seed liquor is obtained.
It is sole carbon source and the liquid MSM of nitrogen source as simulated wastewater to prepare using 100mg/L triazoles, by above-mentioned seed liquor
Add in simulation triazole waste water, with 180 revs/min of rotating speed shaking table culture under the conditions of 30 DEG C, observe in degradation process in waste water
Triazole concentration, observe bacterial growth situation.Set up the blank control for not being inoculated with NJUST43.As shown in Figure 1, the nitrogen of 100mg/L tri-
Azoles can be after being tamed in 9d triazole special efficacy degradation bacteria it is degradable.And it be not inoculated with NJUST43 control sample, three nitrogen
Azoles does not obtain obvious degradation.
This example demonstrates that isolated Bacillus sp.NJUST43 can be sole carbon source and nitrogen using triazole
Source carries out growth and breeding, and can realize the degradable of triazole.
Influence of the triazole concentration of embodiment 2 to triazole degradation property
Prepare the liquid MSM that triazole concentration is respectively 50mg/L, 100mg/L, 150mg/L and 200mg/L and be used as simulation
Waste water, Bacillus sp.NJUST43 seed liquors are accessed into simulated wastewater with 2% inoculum concentration.As shown in Figure 1, Bacillus
Sp.NJUST43 can realize the degradable of triazole in simulated wastewater of the triazole at concentrations up to 150mg/L.It is dense in triazole
Spend under conditions of 50mg/L, 100mg/L and 150mg/L, triazole in simulated wastewater can be realized in 7d, 12d and 16d respectively
Complete removal, triazole can not effectively be degraded under conditions of triazole concentration reaches 200mg/L.Due to triazole
High toxicity and difficult degradation characteristic, under a high concentration condition, although the degradable of triazole can be realized, bacterial growth lag phase
It is obviously prolonged.
This example demonstrates that although Bacillus sp.NJUST43 can realize the degraded of higher concentration triazole, handle
Efficiency has much room for improvement.
Influence of the embodiment 3pH values to triazole degradation property
It is 100mg/L to prepare triazole concentration, and initial pH is 6.0,7.0 and 8.0 triazole simulated wastewater, will
Bacillus sp.NJUST43 seed liquors access simulated wastewater with 2% inoculum concentration.As shown in Figure 2, it is 6.0- in initial pH
Under conditions of 8.0, Bacillus sp.NJUST43 can realize the high efficiency degraded of triazole；Under conditions of initial pH is 7,
Bacillus sp.NJUST43 can also realize that triazole is degradable faster,；And under conditions of being 6.0 and 8.0 in pH, three
The degraded of nitrogen azoles is extremely slow, up in the time of 14 days, it is impossible to which degraded is complete.
This example demonstrates that the optimal pH condition of Bacillus sp.NJUST43 degraded triazole is neutrality；In pH
Under conditions of 7, triazole degradation rate is of a relatively high；The pH conditions of meta-acid and meta-alkalescence are all unfavorable for the degraded of triazole.
Influence of the presence of 4 degradable organic carbon source of embodiment to triazole degradation property
Prepare triazole concentration be 100mg/L, additional degradable organic carbon source (glucose) concentration be respectively 500mg/L,
1000mg/L and 1500mg/L simulated wastewater, Bacillus sp.NJUST43 seed liquors are accessed into simulation with 2% inoculum concentration
Waste water.As shown in Figure 3, lifting of the addition of 500mg/L glucose for the degradation rate of triazole is most obvious；And 1000mg/
L, the addition of 1500mg/L glucose has then delayed the degraded of triazole.
This example demonstrates that the presence of the degradable carbon source of low concentration has the growth beneficial to Bacillus sp.NJUST43,
So as to promote the degradation process of triazole；The presence of the degradable carbon source of high concentration consumes oxygen and nutrient, to three
The degraded of nitrogen azoles produces Reverse transcriptase.
Sequence table
<110>Hubei Zhen Run environmental science and technology limited company
<120>Triazole degraded bacillus and its application
<130> 2017
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1311
<212> DNA
<213> Bacillus sp.
<400> 1
tgacgggcgg tgtgtacaag gcccgggaac gtattcaccg cggcatgctg atccgcgatt 60
actagcgatt ccagcttcat gtaggcgagt tgcagcctac aatccgaact gagaacggtt 120
ttatgagatt agctccacct cgcggtcttg cagctctttg taccgtccat tgtagcacgt 180
gtgtagccca ggtcataagg ggcatgatga tttgacgtca tccccacctt cctccggttt 240
gtcaccggca gtcaccttag agtgcccaac ttaatgatgg caactaagat caagggttgc 300
gctcgttgcg ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac 360
ctgtcactct gctcccgaag gagaagccct atctctaggg ttttcagagg atgtcaagac 420
ctggtaaggt tcttcgcgtt gcttcgaatt aaaccacatg ctccaccgct tgtgcgggcc 480
cccgtcaatt cctttgagtt tcagccttgc ggccgtactc cccaggcgga gtgcttaatg 540
cgttaacttc agcactaaag ggcggaaacc ctctaacact tagcactcat cgtttacggc 600
gtggactacc agggtatcta atcctgtttg ctccccacgc tttcgcgcct cagtgtcagt 660
tacagaccag aaagtcgcct tcgccactgg tgttcctcca tatctctacg catttcaccg 720
ctacacatgg aattccactt tcctcttctg cactcaagtc tcccagtttc caatgaccct 780
ccacggttga gccgtgggct ttcacatcag acttaagaaa ccacctgcgc gcgctttacg 840
cccaataatt ccggataacg cttgccacct acgtattacc gcggctgctg gcacgtagtt 900
agccgtggct ttctggttag gtaccgtcaa ggtgccagct tattcaacta gcacttgttc 960
ttccctaaca acagagtttt acgacccgaa agccttcatc actcacgcgg cgttgctccg 1020
tcagactttc gtccattgcg gaagattccc tactgctgcc tcccgtagga gtctgggccg 1080
tgtctcagtc ccagtgtggc cgatcaccct ctcaggtcgg ctacgcatcg ttgccttggt 1140
gagccgttac ctcaccaact agctaatgcg acgcgggtcc atccataagt gacagccgaa 1200
gccgcctttc aatttcgaac catgcagttc aaaatattat ccggtattag ccccggtttc 1260
ccggagttat cccagtctta tgggcaggtt acccacgtgt tactcacccg t 1311
Claims (3)
- The bacillus 1. triazole is degraded, is Bacillus sp.NJUST43, deposit number is CCTCC NO：M 2017102.
- 2. application of the triazole degraded bacillus according to claim 1 in wastewater treatment containing triazole.
- 3. application according to claim 2, it is characterised in that specific method is：By nitrogen azoles degraded bacillus Bacillus sp.NJUST43 seed liquors are inoculated into waste water containing triazole, culture pH be 6.0~8.0, cultivation temperature be 25~ 35℃。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711249249.6A CN107858309B (en) | 2017-12-01 | 2017-12-01 | Bacillus for degrading triazole and application thereof |
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| CN201711249249.6A CN107858309B (en) | 2017-12-01 | 2017-12-01 | Bacillus for degrading triazole and application thereof |
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| CN107858309B CN107858309B (en) | 2020-11-27 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119167A (en) * | 2016-07-07 | 2016-11-16 | 浙江省农业科学院 | A kind of utilize the method for Triadimenol in novel microorganism bacterial strain degraded soil |
| WO2016196681A1 (en) * | 2015-06-02 | 2016-12-08 | Koch Agronomic Services, Llc | Microbial inoculant compositions and uses thereof in agriculture |
-
2017
- 2017-12-01 CN CN201711249249.6A patent/CN107858309B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016196681A1 (en) * | 2015-06-02 | 2016-12-08 | Koch Agronomic Services, Llc | Microbial inoculant compositions and uses thereof in agriculture |
| CN106119167A (en) * | 2016-07-07 | 2016-11-16 | 浙江省农业科学院 | A kind of utilize the method for Triadimenol in novel microorganism bacterial strain degraded soil |
Non-Patent Citations (2)
| Title |
|---|
| SALUNKHE VP等: "Enhanced dissipation of triazole and multiclass pesticide residues on grapes after foliar application of grapevine-associated Bacillus species.", 《J AGRIC FOOD CHEM》 * |
| 王鑫华等: "氧化塘工艺处理苯并三氮唑生产废水 ", 《工业用水与废水》 * |
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