CN101475925B - A strain of quinoline-degrading bacterium, as well as cultivation method and use thereof - Google Patents
A strain of quinoline-degrading bacterium, as well as cultivation method and use thereof Download PDFInfo
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- CN101475925B CN101475925B CN2009100766683A CN200910076668A CN101475925B CN 101475925 B CN101475925 B CN 101475925B CN 2009100766683 A CN2009100766683 A CN 2009100766683A CN 200910076668 A CN200910076668 A CN 200910076668A CN 101475925 B CN101475925 B CN 101475925B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 23
- 238000012364 cultivation method Methods 0.000 title 1
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Abstract
The present invention relates to a quinoline degrading bacterium and it culture method and applications. The strain is Pseudomonas putida KT-ql-116 CGMCC No.2790, which can effectively degrade high concentration of quinoline compounds (the highest available concentration is 850mg / L), and also can use benzene, toluene, xylene, phenol and other aromatic compounds. The strain can have a very good growth in the coking wastewater, and can effectively degrade quinoline therein, to develop the corresponding environmental protection biological preparation, thereby showing good application prospects.
Description
Technical field
The present invention relates to the new bacterial strain of Rhodopseudomonas, the cultural method of this bacterial strain and the application of this bacterial strain in organic pollutant degradation.
Background technology
Quinoline is typical case's representative of nitrogen-containing heterocycle compound, and purposes is extremely extensive, is important source material and solvent during multiple medicine, agricultural-food, dyestuff etc. are produced.Quinoline in the environment is mainly from coal gas, fossil oil processing, coal tar resistates and Wood preservation facility.In addition, quinoline and verivate thereof still are the Persistent organic pollutants composition in the various industrial sewage such as coking chemical waste water, petroleum wastewater, pharmacy waste water.Respectively the organism of 2 kinds of coking chemical waste waters is formed by the scholar and to be analyzed, point out that the content of quinolines is only second to phenol compound and occupies second.Quinoline and verivate thereof can be carcinogenic, teratogenesis, mutagenicity, and are difficult to biological degradation.Make its water-soluble enhancing because quinoline contains 1 very strong nitrogen-atoms of electronegativity, so this compounds more is prone to, and diffusion exists with lasting in environment.Existing investigation finds that the concentration of quinoline near underground water and the soil the creosote polluted place and hydroxyquinoline thereof is up to several mg/L.In addition, quinoline and verivate thereof also are present in the tissue of urban air, tobacco smoke, seawater and fish, therefore seek the method for effectively removing quinoline and have crucial meaning.
The removal method of pollutent is a lot, roughly is divided into physics method, chemical method and biological process.Though it is high that physics method and chemical method are removed efficient, cost is high, easy generation of secondary pollution.Biological treating has advantages such as cost is low, secondary pollution is light, Environmental compatibility is good, has become one of preferred option in the Pollution abatement technology at present.Successful, effective, the suitable factor of decision biochemical processing process, except processing condition and operational administrative, the effect that is used for the functional microorganism colony of processes such as contaminant degradation, conversion also is crucial.Therefore, to separation, the screening of extraordinary mikrobe, the degradation characteristic of hardly degraded organic substance and the research of mechanism have been become the research focus in environmental science, the life science.
Biodegradable research about quinoline begins just that since the nineties in 20th century detailed report has been arranged; Comprise the evaluation or the like of separation, pathways metabolism and the metabolic gene of degradation bacteria strains; But; Because quinoline itself belongs to the difficult degradation organic heterocyclic molecule, occurring in nature is difficult to find the mikrobe that can effectively degrade to it, thus so far various countries will quinoline as in addition primary study and processing of biodegradable organic compounds; Hope to find some effective mikrobes that it is controlled, thereby guarantee that quinoline content is unlikely to environment is polluted in the trade effluent of various dischargings.
Summary of the invention
First purpose of the present invention provide a strain effectively the degradable organic pollutant quino-be used for the new bacterial strain of environmental pollution improvement.
The new bacterial strain that is used for environmental pollution improvement provided by the present invention is pseudomonas putida (Pseudomonas putida) KT-ql-116; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 9th, 2008 and (abbreviate CGMCC as; The address is in Institute of Micro-biology of the Chinese Academy of Sciences), deposit number is CGMCC No.2790.
Pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 can and add on the inorganic salt screening culture medium of quinoline (100mg/L) at common bacteria LB substratum and grows, and bacteria colony white, rounded is smooth, moistening, and metalluster is arranged.It is G that opticmicroscope is observed it down
-Bacillus extremely gives birth to many flagellums.Obligate is aerobic, respiratory metabolism; Decomposition glucose, wood sugar, oxidase positive.Said inorganic salt screening culture medium can be MM2 substratum: Na
2HPO
42g/L, KH
2PO
41g/L, MgSO
47H
2O 0.1g/L, liquid microelement 5.0ml/L; Wherein every 1000ml liquid microelement comprises CaCl
22H
2O 0.0004g, FeSO
47H
2O 0.04g, MnSO
44H
2O 0.04g, ZnSO
47H
2O 0.02g, CuSO
45H
2O 0.005g, CoCl
26H
2O 0.004g, NaCl 1.0g, Na
2MoO
42H
2O 0.005g.
Pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 in containing the LB substratum of 100mg/L quinoline 25 ℃, 200rpm cultivates 18h～24h, and viable count can reach 10
9～10
10Cfu/ml; In the screening culture medium that contains the different concns quinoline 25 ℃, 200rpm cultivates 18h～166h (along with the raising of quinoline concentration, incubation time prolongs), and viable count can reach 10
5～10
7Cfu/ml, quinoline almost are degraded fully.
Second purpose of the present invention provides a kind of special cultural method of pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCCNo.2790, this cultural method with coking chemical waste water as its sole carbon source, the energy and nitrogenous source.
The method of cultivation pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 provided by the present invention; Be with the substratum of coking chemical waste water, under aerobic conditions, cultivate as pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790.
Said coking chemical waste water general pH is 7.0-7.5, and COD is 1500～2000mg/L, quinoline content 70～110mg/L.
Culture temperature in the said cultural method can be room temperature (about 25 ℃).
Incubation time in the said cultural method can be 3～5 days.
Pseudomonas putida of the present invention (Pseudomonas putida) KT-ql-116 CGMCC No.2790 is effective degrading high concentration quinoline (being up to 850mg/L) not only; This most degradation concentration (500mg/L) than any strain of quinoline-degrading bacterium that document has been reported is all high a lot; But also aromatics such as degradable benzene,toluene,xylene, phenol; Experiment shows; The prolongation along with incubation time in containing the inorganic salt liquid substratum of the above quinoline of 500mg/L of this bacterial strain can make nutrient solution produce pink colour, dark brown or light brown in succession, and there is the generation of different intermediate products in each stage that is illustrated in degraded.Bacterial strain also has good degraded genetic stability to quinoline, in not containing the LB substratum of quinoline, goes down to posterity 5 times, and the last bacterium of going down to posterity is inoculated in the MM2 minimal medium that contains the 500mg/L quinoline, when 44h, can thoroughly degrade quinoline.Experiment shows that pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 coking chemical waste water also capable of using is grown as its sole carbon source, the energy, nitrogenous source.Therefore, the present invention provides pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 to have to be used to the biological treating of the contaminate environment that contains the high density quinoline or the great potential of biological prosthetic.
Based on These characteristics; Pseudomonas putida of the present invention (Pseudomonas putida) KT-ql-116 CGMCCNo.2790 can be used for the improvement of organic pollutant in the environment; The quinolines of particularly effectively degrading; And can develop corresponding environment friendly biological preparation whereby, have higher research and using value.
The preservation of biomaterial
Pseudomonas putida of the present invention (Pseudomonas putida) KT-ql-116 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 9th, 2008 and (abbreviates CGMCC as; The address is Institute of Microorganism, Academia Sinica in the Datun Road, Chaoyang District, Beijing City), deposit number is CGMCC No.2790.
Description of drawings
The quinoline-degrading curve of Fig. 1 pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 in MM2 degraded substratum.
The growth curve of Fig. 2 pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 in coking chemical waste water reaches the degradation curve to quinoline.
Embodiment
Through will more describing the present invention in detail by following examples, but should be understood that following examples only are illustrative, the present invention does not receive the restriction of these embodiment.
Following experimental technique is ordinary method if no special instructions, and the solvent in all substratum is water.
MM2 substratum: Na
2HPO
42g/L, KH
2PO
41g/L, MgSO
47H
2O 0.1g/L, liquid microelement 5.0ml/L; Wherein every 1000ml liquid microelement comprises CaCl
22H
2O 0.0004g, FeSO
47H
2O 0.04g, MnSO
44H
2O 0.04g, ZnSO
47H
2O 0.02g, CuSO
45H
2O 0.005g, CoCl
26H
2O 0.004g, NaCl 1.0g, Na
2MoO
42H
2O 0.005g.
The detection method of quinoline adopts performance liquid chromatography (HPLC) method:
Sample preparation: in the aerobic degradation process, sampling at regular intervals, water sample is centrifugal 10min under 8000rpm, and supernatant is used for the HPLC quantitative analysis of quinoline behind 0.45 μ m membrane filtration.
Chromatographic instrument is Tianjin, island LC-20A, and chromatographic column is the reverse post of Dalian Yi Lite C18,250mm * 4.6mm, and granularity is 5 μ m; Moving phase is 60% (v/v) methanol aqueous solution, and flow rate of mobile phase is 1.0ml/min.Measure wavelength 230nm, about RT 6min.
Separation, purifying and the evaluation of embodiment 1, pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790
Pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 separates in the sludge sewage of Xiao Jia river, Beijing, and concrete enrichment, separation, purge process are following:
The sample of gathering is inoculated in the MM2 isolation medium that contains quinoline, 25 ℃, 180rpm vibration down, quinoline concentration increases to 100mg/L gradually from initial 30mg/L, increases the interpolation concentration of a quinoline during this weekly.From the pregnant solution of domestication, get the 1ml nutrient solution then and join (liquid amount is the 20ml/100ml triangular flask) in the MM2 liquid nutrient medium that contains the 300mg/L quinoline, put 200rpm cultivation in 25 ℃ of shaking tables.Find after 7 days that the nutrient solution pulverize is red, show that quinoline is degraded under the effect of mud, generate coloured intermediate product, this moment the bacterium in the nutrient solution is carried out coated plate and separate.Nutrient solution is diluted 10 respectively
5With 10
4Doubly; Drawing 100 μ l is applied on the MM2 minimal medium flat board that contains the 100mg/L quinoline; Put in 25 ℃ of constant incubators and cultivate; Until growing obvious visible bacterium colony, picking list bacterium colony is scoring on new MM2 minimal medium (the containing the 100mg/L quinoline) flat board and carries out purifying, obtains well-grown single bacterium colony at last.The bacterial strain that obtains is carried out morphologic observation, Physiology and biochemistry evaluation and 16SrRNA gene sequencing to be identified.The result shows that this bacterial strain can add on quinoline (100mg/L) screening culture medium at common bacteria LB substratum and inorganic salt and grows, and bacteria colony white, rounded is smooth, moistening, and metalluster is arranged.It is G that opticmicroscope is observed it down
-Bacillus extremely gives birth to many flagellums.Obligate is aerobic, respiratory metabolism; Decomposition glucose, wood sugar, oxidase positive.After the compare of analysis of the partial sequence of 16SrRNA gene is pseudomonas putida (Pseudomonas putida) with this identification of strains.With this bacterial strain called after pseudomonas putida (Pseudomonas putida) KT-ql-116.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 9th, 2008, and deposit number is CGMCC No.2790.
Embodiment 2, pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 compose the degraded of pollutent
MM2 is as minimum medium, with the energy, carbon source or the nitrogenous source of the organic pollutant shown in the table 1 as unique interpolation, adopts the mode of solid culture, 25 ℃, leave standstill and cultivate a week, observe the situation that bacterium colony forms.Test result to compounds such as benzene, phenol, toluene, YLENE, pyridine, prussiate, quinoline, imidazoles is as shown in table 1.The result shows; The degraded of pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 spectrum is still than broad; Can utilize all aromatics to be tried, for nitrogen-containing heterocycle compound, especially quinoline had higher degrading activity.
Table 1KT-ql-116 utilizes situation to the degraded of organic pollutant
| Pyridine | YLENE | Benzene | Phenol | Toluene | Quinoline | CN - | Imidazoles | |
| Concentration | 1.96g/L | 1.72g/L | 4.39g/L | 50mg/L | 1.72g/L | 0.5g/L | 200mg/L | 50g/L |
| Growing state | - | ++ | + | ++ | ++ | √ | - | - |
Annotate: "+" expression thalline is grown on corresponding pollutent flat board in the table, and "+" multilist more shows that bacterium looks good more; "-" expression thalline is not long on corresponding pollutent flat board, and the growth on corresponding pollutent flat board of " √ " expression thalline is vigorous, and pollutent has no restraining effect to bacterium.
Embodiment 3, pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 are to the biological degradation of quinoline
1) mensuration of pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 quinoline-degrading curve
Thalline grows to the righttest cell age in containing the LB enrichment medium of 100mg/L quinoline; 4000rpm, 10min are centrifugal, washing, collect, and are connected to then in the degraded substratum (MM2), and the quinoline starting point concentration is about 260mg/L; Sampling at regular intervals; Measure the quinoline residual concentration, draw out degradation curve, the result sees Fig. 1.Visible by Fig. 1, under the condition of resting cell, just can the quinoline about 260mg/L almost be degraded fully in the thalline 2h, the efficient of visible strains for degrading quinoline is very high.
2) pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 is to the tolerance of different concns quinoline and the examination of degradation capability
Choosing a ring lawn from the preservation flat board of bacterial strain is inoculated in the MM2 liquid nutrient medium that contains the different concns quinoline; Quinoline concentration is respectively 500mg/L, 650mg/L, 700mg/L, 750mg/L, 800mg/L, 850mg/L, 900mg/L, 25 ℃; 200rpm, shaking culture.The result finds, removes under the situation of 900mg/L, and thalline is beyond the growth, waits to try in the substratum all breeding in various degree at other, and it is muddy that nutrient solution becomes.When thalline is grown 40.5h, 64h, 124h, 142h, 153h and 166h under quinoline concentration is respectively the situation of 500mg/L, 650mg/L, 700mg/L, 750mg/L, 800mg/L and 850mg/L; Bacterium liquid OD600 can reach 0.7～0.8; Quinoline under all situations all almost is degraded fully at this moment, and finds that nutrient solution all becomes dark brown or light brown.All results show, thalline can be sole carbon source, nitrogenous source and energy growth with the quinoline, and the concentration that can well tolerate and degrade is up to the quinoline of 850mg/L, and well propagation takes place under this concentration, and the higher viable count of living weight can reach 10
6～10
7Cfu/ml.This result shows that pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 has good application prospects.
3) examination of the genetic stability of pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 degraded quinoline
Bacterial strain KT-ql-116 is gone down to posterity 5 times in not containing the LB substratum of quinoline; The last bacterium of going down to posterity is inoculated in the MM2 minimal medium that contains the 500mg/L quinoline; When 44h, can thoroughly degrade quinoline, show that this bacterial strain has good genetic stability to the degraded of quinoline.This characteristic is very important to production application.
Embodiment 4, the application of pseudomonas putida (Pseudomonas putida) KT-ql-116 CGMCC No.2790 in Treatment of Coking Effluent
Choose two ring bacterium accesses from the LB substratum of preservation strain and be equipped with the 300ml triangular flask of 30ml LB liquid nutrient medium, 25 ℃, 200rpm, shaking culture is spent the night.After treating that thalline gets into logarithmic phase, draw the access of 1ml bacterium liquid and be equipped with in the 300ml triangular flask of 30ml through the Shoudu Iron and Steel Co coking chemical waste water of filtering bacterium, 25 ℃, 200rpm, shaking culture 5d surveys OD600 at regular intervals one time, draws out growth curve at last; In addition, the residual quantity of sampling and measuring quinoline is drawn degradation curve simultaneously at regular intervals, and the result is as shown in Figure 2.Find out that by Fig. 2 pseudomonas putida (Pseudomonas putida) KT-ql-116CGMCC No.2790 can utilize the coking chemical waste water of Shoudu Iron and Steel Co to grow preferably, breed.The preceding 48h that thalline is grown in coking chemical waste water is in lag phase basically, and cell concentration is lower; 48h～72h gets into logarithmic phase, and cell concentration increases very soon, and this moment, cell concn can reach 10
9Cfu/ml, OD600=1.6; Begin behind the 72h to get into stationary phase, cell concentration is constant basically.And the quinoline in the waste water does not almost have obvious variation at preceding 48h, and when thalline began to get into logarithmic phase, quinoline also along with beginning degraded, was promptly degraded in the quinoline 20h in logarithmic phase fully.This shows pseudomonas putida (Pseudomonasputida) though KT-ql-116 CGMCC No.2790 grows slower in coking chemical waste water; But thalline can reach very high living weight in 72h; And can the quinoline in the waste water be degraded fully; Explain that pseudomonas putida (Pseudomonasputida) KT-ql-116 CGMCC No.2790 can well grow in coking chemical waste water, and be expected to be used for the biochemical treatment of coking chemical waste water.
Claims (5)
1. a pseudomonas putida (Pseudomonas putida) KT-ql-116, its preservation center registration number is CGMCC No.2790.
2. preservation center registration number is the cultural method of pseudomonas putida (Pseudomonas putida) KT-ql-116 of CGMCC No.2790; With coking chemical waste water as its substratum; Under aerobic conditions, cultivate; The pH of wherein said coking chemical waste water is 7.0-7.5, and COD is 1500～2000mg/L, quinoline content 70～110mg/L.
3. cultural method as claimed in claim 2 is characterized in that: culture temperature is a room temperature, and incubation time is 3～5 days.
4. preservation center registration number is the application of pseudomonas putida (Pseudomonas putida) KT-ql-116 in curbing environmental pollution of CGMCC No.2790, and it is characterized in that, utilizes the quinoline in this bacterium degraded environment.
5. preservation center registration number is that pseudomonas putida (Pseudomonas putida) KT-ql-116 of CGMCC No.2790 is in preparation be used for the degrading application of biotechnological formulation of quinoline.
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