CN106119159B - The comamonas of one high-efficiency degradation pyridine carboxylic acid and its application - Google Patents
The comamonas of one high-efficiency degradation pyridine carboxylic acid and its application Download PDFInfo
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- CN106119159B CN106119159B CN201610497030.7A CN201610497030A CN106119159B CN 106119159 B CN106119159 B CN 106119159B CN 201610497030 A CN201610497030 A CN 201610497030A CN 106119159 B CN106119159 B CN 106119159B
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Abstract
The present invention provides the comamonas of a high-efficiency degradation pyridine carboxylic acid and its applications, belong to technical field of bioengineering.The present invention separates one plant of pyridine carboxylic acid degradation bacteria, comamonas, and classification naming is Comamonas sp., is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.10700.The pyridine carboxylic acid it is an advantage of the invention that comamonas can effectively degrade in the higher situation of the pH value of wider range, especially basicity；The comamonas can be administered as biological reinforced formulation application in the aerobe of weak base pyridine carboxylic acid waste water into weakly acidic condition, be had broad application prospects.
Description
Technical field
The invention belongs to technical field of bioengineering, and in particular to the comamonas of a high-efficiency degradation pyridine carboxylic acid and
It is applied.
Background technique
Pyridine carboxylic acid and its derivative are widely used in the production of medicine, pesticide and household chemicals, pyridine carboxylic acid
Extensive use causes it to pollute environment.Pyridine carboxylic acid waste water can be handled by aerobe method.Currently, aerobic degradation pyridine
The microbial resources of formic acid mainly have arthrobacterium, mycobacteria and streptomycete etc..It is still few about other bacterial strain reports for belonging to kind
See.In addition, the pyridine carboxylic acid degradation bacteria that document is reported all is grown within the scope of comparatively gentle pH, substantially 6.0 to 7.0
In range, and practical pyridine carboxylic acid industrial wastewater pH variation range is wider, therefore the pyridine carboxylic acid degradation bacteria that document is reported is real
Border application is restricted.
Summary of the invention
The purpose of the present invention is to provide the comamonas of a high-efficiency degradation pyridine carboxylic acid and its application, can compared with
Pyridine carboxylic acid is efficiently removed in wide ph range.
In order to achieve the above objectives, the technical solution adopted by the present invention are as follows:
The comamonas of one high-efficiency degradation pyridine carboxylic acid, classification naming are Comamonas sp., are preserved in China
Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC NO.10700.
Application of the comamonas of the efficient degradation pyridine carboxylic acid in terms of pyridine carboxylic acid of degrading.
The comamonas of the efficient degradation pyridine carboxylic acid is in terms of preparing biological bacteria preparation or biological reinforced preparation
Application.
The comamonas of the efficient degradation pyridine carboxylic acid is as biological bacteria preparation or biological reinforced preparation in pyridine
Application in formic acid wastewater processing.
Under conditions of pyridine carboxylic acid is sole carbon source, nitrogen source and the energy, pH 7, temperature are 30 DEG C, comamonas energy
It is enough to degrade to pyridine carboxylic acid concentration≤800mg/L waste water.
Under conditions of pyridine carboxylic acid is sole carbon source, nitrogen source and the energy, temperature is 30 DEG C, pH is 5~9, Comamonas
Bacterium can degrade to pyridine carboxylic acid concentration≤200mg/L waste water.
Compared with the existing technology, the invention has the benefit that
The present invention the pyridine carboxylic acid degradation bacteria that previous literature is reported there are aiming at the problem that, pass through scientific method domestication, sieve
One plant of new pyridine carboxylic acid degradation bacteria-comamonas is selected, classification naming is Comamonas sp., and it is micro- to be preserved in China
Biological inoculum preservation administration committee common micro-organisms center, deposit number are CGMCC NO.10700.The comamonas can be
Pyridine carboxylic acid is efficiently removed within the scope of wider pH value, thus the degradation treatment pyridine first suitable for practical pyridine carboxylic acid industrial wastewater
Acid.Comamonas provided by the invention can be used as biological bacteria preparation and be added in existing pyridine carboxylic acid waste water treatment system,
Enhance the processing capacity of former processing system；Comamonas provided by the invention can be as biological reinforced formulation application in weak base
Into weakly acidic condition, the aerobe of pyridine carboxylic acid waste water is administered, and has wide application latent in the processing of pyridine carboxylic acid waste water
Power.
Further, it is found through experiments that, comamonas provided by the invention is in pH=7,30 DEG C of temperature, pyridine carboxylic acid
It, being capable of degradable pyridine carboxylic acid under conditions of initial concentration≤800mg/L；In addition, being 5~9 in pH, pyridine carboxylic acid is unique
Carbon, nitrogen, the energy, 30 DEG C of temperature, and under conditions of pyridine carboxylic acid initial concentration≤200mg/L, which can be complete
Degradation pyridine carboxylic acid.
Preservation explanation
The comamonas of efficient degradation pyridine carboxylic acid of the present invention has carried out following preservations:
The preservation time: on April 9th, 2015, preservation place: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the micro- life of China
Object culture presevation administration committee common micro-organisms center, CGMCC；Deposit number is CGMCC NO.10700.
Detailed description of the invention
Fig. 1 is comamonas provided by the invention initial as sole carbon, nitrogen, the energy and pyridine carboxylic acid using pyridine carboxylic acid
Concentration is the colony morphology characteristic in the Solid agar culture of 100mg/L: after cultivating 3 days at 30 DEG C, bacterium colony is rice white, side
Edge is irregular, rough surface is rough, surface does not swell but combines closely in media surface, and difficult in inoculation
To provoke.
Fig. 2 is comamonas provided by the invention using pyridine carboxylic acid as sole carbon, nitrogen, the energy (initial concentration
Fluid nutrient medium 100mg/L) cultivates the scanning electron microscope (SEM) photograph after 10h at 30 DEG C, and cell is rod-short, and length dimension is 1~2 μ
m。
Fig. 3 is comamonas provided by the invention using pyridine carboxylic acid as sole carbon, nitrogen, the energy, pH 7, temperature 30
DEG C, pyridine carboxylic acid mass concentration be 800mg/L fluid nutrient medium in growth with pyridine carboxylic acid degradation curve figure.
Fig. 4 be comamonas provided by the invention at various ph values, 30 DEG C of temperature, pyridine carboxylic acid be sole carbon, nitrogen,
When the energy and pyridine carboxylic acid initial concentration are 200mg/L, the degradation curve figure of pyridine carboxylic acid.
Specific embodiment
Below with reference to technical solution and attached drawing, the present invention will be described in further detail.
1, comamonas provided by the invention, screening step are as follows:
It is collected oxygen activity mud sample from the aeration tank of Xi'an Medicine Factory, tamed, cultivated as bacterium source.It will
50mL activated sludge, the sodium pyrophosphate for being added 0.2% are used as solution flocculant, vibrate 5~10min on the oscillator and smash active dirt
After mud wadding body, 5mL activated sludge is taken, is added in the shaking flask of the minimal medium containing 95mL, adds pyridine carboxylic acid as unique
Carbon, nitrogen, the energy, wherein the concentration of pyridine carboxylic acid is 100mg/L, carries out shaking table culture, 30 DEG C of temperature, revolving speed 180r/min.Nothing
Machine salt culture medium ingredient: Na2HPO4·12H2O, 7g/L；KH2PO4, 1g/L；CaCl2·2H2O, 10mg/L；FeCl3, 2mg/L；
MgSO4·7H2O, 20mg/L.When bacterium solution becomes muddy, pyridine carboxylic acid can't detect, be transferred next time；By constantly taming and dociling
Change culture, finally obtains using pyridine carboxylic acid as the stabilization bacterium solution of sole carbon, nitrogen, the energy.Bacterium solution dilution will be stablized, with pyridine first
Acid is sole carbon, is coated on the solid medium of nitrogen, the energy.Solid culture based component: Na2HPO4·12H2O, 7g/L；
KH2PO4, 1g/L；CaCl2·2H2O, 10mg/L；FeCl3, 2mg/L；MgSO4·7H2O, 20mg/L；Pyridine carboxylic acid, 100mg/L；
Agar 2%.The solid medium is put into illumination box, 30 DEG C, culture 4~5 days choose single colonie；Separate one
Strain using pyridine carboxylic acid as sole carbon, nitrogen, the energy comamonas；The bacterial strain identified by 16S rDNA sequence, in
On April 9th, 2015 is preserved in " China Committee for Culture Collection of Microorganisms's common micro-organisms center ", and deposit number is
CGMCC No.10700, classification naming are Comamonas sp..Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postal
It compiles: 100101.
Growth pH wider range of the comamonas, there is preferable growth within the scope of 5.0~9.0 pH, most suitable
PH value is 7.0；Its morphological feature: using pyridine carboxylic acid as sole carbon, nitrogen, the energy fluid nutrient medium, cultivate 10h at 30 DEG C
Afterwards, as shown in Fig. 2, cell is rod-short, length dimension is 1~2 μm；Using pyridine carboxylic acid as sole carbon, nitrogen, the energy agar
On solid medium, after being cultivated 3 days at 30 DEG C, as shown in Figure 1, bacterium colony be rice white, edge be irregular, rough surface not only
Sliding, surface does not swell but combines closely in media surface, and is difficult to provoke in inoculation.The comamonas was both
It can be grown in eutrophy culture medium LB, can also be the inorganic of sole carbon source in small organic molecules such as glucose, sucrose, starch
It is grown in salt culture medium.
The 16S rDNA of comamonas provided by the invention is expanded: the 16S rDNA by expanding the bacterial strain is obtained
Length is the 16S rDNA sequence of 1428bp.PCR primer uses universal primer 27F (the 5 '-AGT TTG ATC of 16S rDNA
MTG GCT CAG-3 ') and 1492R (5 '-GGT TAC CTT GTT ACG ACT T-3 ').With PCR instrument (Applied
Biosystems, 2720thermal cycler) it carries out amplification reaction.Table 1 and 2 respectively illustrates PCR reaction system and anti-
Answer condition.
1 PCR reaction system of table
2 reaction condition of table
1 μ L of PCR product is taken to carry out 1% agarose gel electrophoresis, with DNA QIAquick Gel Extraction Kit (SK8131 plastic recovery kit)
Target fragment is recycled, PCR product is cloned on pGEM-T carrier, converts competent escherichia coli cell.In Genbank into
Row BLAST is compared.With 5.1 software building phylogenetic tree of MEGA, closed using Neighbo-Joining method analysis bacterial strain relationship
It is (Bootstrap value=1000).The Serial No. KP900021 logged in Genbank, with Comamonas
Sp.JC13 homology infers that the bacterial strain belongs to Comamonas (Comamonas) up to 99%.
2, the cellular liquid culture of comamonas is prepared:
Comamonas single colonie of the picking on the solid medium that pyridine carboxylic acid is sole carbon, nitrogen, the energy, loading are gone out
Bacterium using pyridine carboxylic acid as sole carbon, nitrogen, the energy fluid nutrient medium in, wherein the concentration of pyridine carboxylic acid be 100mg/L, in 30
DEG C, pH=7.0,180r/min, carry out aerobic culture 12~30 hours, cultured bacterium solution 1mL taken to be seeded in 100mL containing pyridine
In the minimal medium of formic acid (200mg/L), 30 DEG C, pH=7.0,180r/min, the aerobic culture of progress 20 hours obtain clump
The cellular liquid culture of hair monad.
3, the research about comamonas to pyridine carboxylic acid aerobic degradation, its step are as follows:
[1] 90mL minimal medium is added in 250mL conical flask, adds the pyridine carboxylic acid of 800mg/L as only
One carbon, nitrogen, the energy sterilize at 121 DEG C；
[2] the cellular liquid culture of the 10mL comamonas prepared is added in above-mentioned 90mL culture medium, 30 DEG C,
PH=7,180r/min, aerobic culture, as a result as shown in Figure 3.
Fig. 3 is comamonas provided by the invention using pyridine carboxylic acid as sole carbon, nitrogen, the energy, pH 7, temperature 30
DEG C, pyridine carboxylic acid mass concentration be 800mg/L fluid nutrient medium in growth with pyridine carboxylic acid degradation curve figure, show feathering
The growth of monad and pyridine carboxylic acid are degraded situation, and abscissa h indicates the time in Fig. 3, and unit is hour, and left side ordinate indicates
The concentration of pyridine carboxylic acid, unit mg/L, right side ordinate indicate dry cell weight, unit mg/L；- ■-represents pyridine first
Acid concentration ,- -represent thalli growth amount.From figure 3, it can be seen that comamonas is 7,30 DEG C of temperature, pyridine first in pH
Under conditions of sour mass concentration≤800mg/L can degradable pyridine carboxylic acid, therefore can as biological reinforced preparation to pyrrole
Pyridine formic acid concn≤800mg/L pyridine carboxylic acid waste water is handled.
4, about the comamonas research to pyridine carboxylic acid aerobic degradation at various ph values, its step are as follows:
[1] minimal medium that secure ph is 3,4,5,6,7,9,10,12 respectively, is added the pyridine first of 200mg/L
Acid is used as sole carbon, nitrogen, the energy, sterilizes at 121 DEG C；
[2] the cellular liquid culture of the 10mL comamonas prepared is separately added into the 90mL of above-mentioned different pH value
In culture medium, 30 DEG C, 180r/min, aerobic culture, as a result as shown in Figure 4.
Fig. 4 be comamonas provided by the invention at various ph values, 30 DEG C of temperature, pyridine carboxylic acid be sole carbon, nitrogen,
When the energy and pyridine carboxylic acid initial concentration are 200mg/L, the degradation curve figure of pyridine carboxylic acid shows comamonas in difference
Growth under pH value and pyridine carboxylic acid are degraded situation, and abscissa h indicates the time in Fig. 4, and unit is hour, and ordinate indicates pyridine
The degradation rate of formic acid, unit %.As can be seen from Figure 4: comamonas pH value be 5~9,30 DEG C of temperature, pyridine first
Under conditions of acid concentration≤200mg/L can degradable pyridine carboxylic acid, therefore can as biological reinforced preparation to wider model
The higher pyridine carboxylic acid waste water of the pH value enclosed, especially basicity carries out degradation treatment.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way, it is all according to the present invention
Technical spirit any simple modification, change and equivalent structure transformation to the above embodiments, still fall within skill of the present invention
In the protection scope of art scheme.
Claims (6)
1. the comamonas of a high-efficiency degradation pyridine carboxylic acid, it is characterised in that: its classification naming is Comamonas sp.,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC NO.10700.
2. application of the comamonas of efficient degradation pyridine carboxylic acid described in claim 1 in terms of pyridine carboxylic acid of degrading.
3. the comamonas of efficient degradation pyridine carboxylic acid described in claim 1 is preparing biological bacteria preparation or biological reinforced system
Application in terms of agent.
4. the comamonas of efficient degradation pyridine carboxylic acid described in claim 1 is as biological bacteria preparation or biological reinforced preparation
Application in pyridine carboxylic acid wastewater treatment.
5. application as claimed in claim 4, it is characterised in that: pyridine carboxylic acid be sole carbon source, nitrogen source and the energy, pH 7,
Under conditions of temperature is 30 DEG C, comamonas can degrade to pyridine carboxylic acid concentration≤800mg/L waste water.
6. application as claimed in claim 4, it is characterised in that: pyridine carboxylic acid is sole carbon source, nitrogen source and the energy, temperature is
30 DEG C, pH be 5~9 under conditions of, comamonas can degrade to pyridine carboxylic acid concentration≤200mg/L waste water.
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|---|---|---|---|---|
| CN101070528A (en) * | 2007-06-01 | 2007-11-14 | 广东省生态环境与土壤研究所 | Iron-reduced tuftedmonas and its use |
| CN101250491A (en) * | 2007-10-24 | 2008-08-27 | 大连理工大学 | Streptomycesalbidoflavus having new metabolic characteristics and its use in biological degradation |
| CN103146604A (en) * | 2013-02-27 | 2013-06-12 | 中蓝连海设计研究院 | Comamonas testosteroni LH-N5 and heterotrophic nitrification-aerobic denitrification microbial inoculum, and preparation method and application thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101070528A (en) * | 2007-06-01 | 2007-11-14 | 广东省生态环境与土壤研究所 | Iron-reduced tuftedmonas and its use |
| CN101250491A (en) * | 2007-10-24 | 2008-08-27 | 大连理工大学 | Streptomycesalbidoflavus having new metabolic characteristics and its use in biological degradation |
| CN103146604A (en) * | 2013-02-27 | 2013-06-12 | 中蓝连海设计研究院 | Comamonas testosteroni LH-N5 and heterotrophic nitrification-aerobic denitrification microbial inoculum, and preparation method and application thereof |
Non-Patent Citations (2)
| Title |
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| Microbial metabolism of pyridine, quinoline, acridine, and their derivatives under aerobic and anaerobic conditions;Kaiser JP等;《Microbiol Rev.》;19960930;第60卷(第3期);483-498 * |
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