CN102399714B - Quinoline degrading bacterium and application thereof - Google Patents
Quinoline degrading bacterium and application thereof Download PDFInfo
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- CN102399714B CN102399714B CN 201110312432 CN201110312432A CN102399714B CN 102399714 B CN102399714 B CN 102399714B CN 201110312432 CN201110312432 CN 201110312432 CN 201110312432 A CN201110312432 A CN 201110312432A CN 102399714 B CN102399714 B CN 102399714B
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Abstract
The invention discloses a quinoline degrading bacterium and application thereof. The quinoline degrading bacterium Q2 belongs to bacillus sp and is collected in China Center for Type Culture Collection (CCTCC), wherein the collection date is July 6, 2011; the collection number is CCTCC NO:M2011239; and the GenBank accession number of the bacterial strain 16S ribosomal deoxyribonucleic acid (rDNA) is JN132107. The quinoline degrading bacterium is a gram-positive bacterium, has a milky white bacterial colony, a tidy edge and a round shape, and is smooth and wet, and convex in the center. The quinoline degrading bacterium can grow and reproduce by utilizing quinoline as the only carbon source, nitrogen source and energy and can completely degrade the quinoline at the concentration of 500 mg/L within 30 hours. The Q2 can tolerate the quinoline at the concentration of 1,400 mg/L, has an excellent degrading effect on high-concentration quinoline industrial wastewater, can be applied to biological enhanced treatment of oil refining wastewater, coking wastewater or tar processing wastewater, and has a wide application prospect.
Description
Technical field
The invention belongs to environmental pollutant biological reinforcing technology field, be specifically related to a strain of quinoline-degrading bacterium and application thereof.
Background technology
Quinoline and its derivates is the fragrant nitrogen-containing heterocycle compound of typical many cyclophanes, extensively is present in pitch, shale oil and crude oil, and be important raw material and the solvents in field such as dyestuff, paint, agricultural chemicals.Quinoline is that a kind of toxicity is large, teratogenecity and the strong organism of carinogenicity, is difficult to biological degradation, has than highly water-soluble and potential mobility, easily by the soil pollution groundwater resource, human health and ecotope is had to huge potential hazard.
With the physics and chemistry method, compare, it is large that biological treatment has treatment capacity, and cost is lower, and mild condition does not produce the characteristics such as secondary pollution, more meets the requirement of the friendly type of constructing environment and conservation-minded society.Yet traditional biologic treating technique has been difficult to meet the environmental requirement of increasingly stringent.In recent years, biological reinforcing technology, on the basis that does not change existing treatment facility, by the interpolation of microorganisms with specific functions, improves the removal usefulness of original biological treatment system to target contaminant, greatly improve sewage treatment capacity, thereby more and more caused people's attention.
At present, existing some scholars filters out the bacterial strain that quinoline is had to the efficient degradation performance both at home and abroad, as pseudomonas (Pseudomonas sp.), Burkholderia pickettii (Burkholderia pickettii), rhodococcus (Rhodococcus sp.), Comamonas (Comamonas sp.), Bacterium lacticum (Lactobacillus), white-rot fungi (white rot fungi), Moraxella (Moraxella sp.), indoles desulfurization bacterium (Desulfobacterium indolicum) etc., these researchs only rest on laboratory stage mostly.Up to the present, there is some scholars research to show that genus bacillus (Bacillus sp.) can degrade 4 both at home and abroad, 5,6-trichlorine hydroxyanisole (4,5,6-trichloroguaiacol), benzo [a] pyrene (benzo[a] pyrene) and pyridine (pyridine), but have no the report of its degraded quinoline.Contain certain density quinoline in refinery water, coking chemical waste water and tar processing waste water, and quinoline belongs to the bio-refractory organism, therefore only depend on the common micro-organisms in biological treatment device to be difficult to realize the effective degraded to quinoline.
Summary of the invention
The present invention is intended to solve common micro-organisms to the inefficient problem of quinoline-degrading, and purpose is to provide a strain of quinoline-degrading bacterium, realizes that it is to refinery water, coking chemical waste water and the biological reinforced processing of tar processing waste water.
Quinoline-degrading bacterium Q2 provided by the present invention comes from Wuhan Branch, Sinopec Corp.'s on-site and is subject to the petroleum chemicals contaminated soil.The quinoline of usining carries out growth and breeding as sole carbon source, nitrogenous source and the energy, in the quinoline minimal medium, the flora in soil is tamed to cultivation, and obtains by the plate streaking separation and purification.
Wherein, quinoline minimal medium composition is: Na
2hPO
412H
2o 4.26g, KH
2pO
42.65g, MgSO
47H
2o 0.20g, CaCl
20.006g, FeSO
47H
2o 0.02g, 2mL trace element storing solution, supply distilled water to 1000mL; Trace element storing solution component is MnSO
4h
2o 0.1g, ZnSO
47H
2o0.12g, H
3bO
30.07g, Na
2moO
4h
2o 0.04g, CuSO
45H
2o 0.02g, CoCl
20.04g, supply distilled water to 1000mL.
This bacterium is gram-positive microorganism, be accredited as genus bacillus (Bacillus sp) through 16S rRNA, numbering Q2, the GenBank accession number of its 16S rDNA is JN132107, be preserved in Chinese Typical Representative culture collection center (Wuhan University) on July 6th, 2011, deposit number is CCTCC NOM2011239.Physio-biochemical characteristics are identified and are shown that this bacterium is hydrogen sulfide production test, V-P test and the nitrate reduction test positive, indole test, methyl red test and Starch Hydrolysis negative.Bacterium colony is oyster white, and neat in edge is rounded, smooth moistening, central protuberance.
More excellent culture condition: temperature is 30 ℃, and initial pH is 7～10, and inoculum size is 20%, and shaking speed is 100rpm.
The invention still further relates to the application of described genus bacillus (Bacillus sp) Q2 in quinoline-degrading, be applied to the quinoline in the biological degradation trade effluent, trade effluent is a kind of in refinery water, coking chemical waste water and tar processing waste water.
Compared with prior art, the genus bacillus of invention (Bacillus sp) Q2 can utilize quinoline to carry out growth and breeding as sole carbon source, nitrogenous source and the energy, in 30h, that the quinoline-degrading of 500mg/L is complete.Genus bacillus (Bacillus sp) Q2 can tolerate the quinoline of concentration up to 1400mg/L, higher concentration quinoline trade effluent is had to good degradation effect, this bacterium can be applicable to the biological reinforced processing of refinery water, coking chemical waste water and tar processing waste water, has a extensive future.
The accompanying drawing explanation
Growth and quinoline-degrading effect relation figure that Fig. 1 is genus bacillus (Bacillus sp) Q2;
Fig. 2 is the impact of quinoline starting point concentration on genus bacillus (Bacillus sp) Q2 degraded quinoline;
Fig. 3 is the impact of culture temperature on genus bacillus (Bacillus sp) Q2 degraded quinoline;
Fig. 4 is the impact of initial pH value on genus bacillus (Bacillus sp) Q2 degraded quinoline;
Fig. 5 is the impact of inoculum size on genus bacillus (Bacillus sp) Q2 degraded quinoline;
Fig. 6 is the impact of shaking speed on genus bacillus (Bacillus sp) Q2 degraded quinoline;
Fig. 7 is the intensifying treatment effect of genus bacillus (Bacillus sp) Q2 to the refinery water of interpolation higher concentration quinoline;
Fig. 8 is the intensifying treatment effect of genus bacillus (Bacillus sp) Q2 to the coking chemical waste water of interpolation higher concentration quinoline;
Fig. 9 is the intensifying treatment effect of genus bacillus (Bacillus sp) Q2 to the tar processing waste water of interpolation higher concentration quinoline.
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention being described further, is not the restriction to its protection domain.
Embodiment 1: the separation of genus bacillus (Bacillus sp) Q2, evaluation and to the degradation property of quinoline
1, the separation of bacterial strain, purifying
(1) bacterial classification source
Contaminated soil in Wuhan Branch, Sinopec Corp.'s sewage works is picked up from the bacterium source, is chocolate, gathers apart from 4 parts of the pedotheques of top layer 3～6cm.
(2) separation and purification of bacterial strain
The soil sample of collection is added to the sterile distilled water sky 24h that exposes to the sun, get the 10mL supernatant liquor after standing to be inoculated in 90mL containing in the LB substratum (peptone 1%, sodium-chlor 1%, yeast extract 0.5%) of 200mg/L quinoline, at 30 ℃ and 150r/min activation culture 24h.Getting the 5mL nutrient solution is forwarded in the quinoline minimal medium containing quinoline 200mg/L, 2～3d is cultivated in the similarity condition domestication, then get the 5mL nutrient solution and move and be connected to fresh quinoline minimal medium, 3 times so repeatedly, wherein quinoline concentration be respectively 200,500,1000mg/L; To press the different ratios dilution containing the domestication liquid of quinoline 1000mg/L, coat respectively on the quinoline inorganic salt flat board containing quinoline 500mg/L, cultivate 4d for 30 ℃.Select sharp-edged bacterium colony, through the LB plate streaking, be purified to and obtain single bacterium colony, carry out respectively the quinoline-degrading test.After determining each strains for degrading quinoline ability, with the good bacterial strain of LB slant preservation degradation property containing quinoline 500mg/L.
2, the evaluation of bacterial strain
(1) colony morphology characteristic of quinoline-degrading bacterial strain Q2 and physio-biochemical characteristics
Quinoline-degrading bacterial strain Q2 bacterium colony is oyster white, and neat in edge is rounded, smooth moistening, central protuberance.
Physio-biochemical characteristics are identified and are shown that this bacterium is hydrogen sulfide production test, V-P test and the nitrate reduction test positive, indole test, methyl red test and Starch Hydrolysis negative.
(2) the 16S rRNA of quinoline-degrading bacterial strain Q2 identifies
16S rRNA gene to quinoline-degrading bacterial strain Q2 is cloned, is checked order, and then in GenBank, carries out the Blast comparison.Result shows, with its sequence similarity reach 99% be bacillus (Bacillus sp), its 16S rDNA sequence is as shown in sequence table.In conjunction with the physio-biochemical characteristics of bacterial strain, it is belonged to bacillus (Bacillus sp), be numbered Q2.
3, the preparation of bacteria suspension
Picking one ring microbionation is in the LB substratum, 30 ℃, 150rpm are cultivated 24h, get the 5mL culture and be connected to fresh LB substratum, cultivate 7～8h (bacterium is in logarithmic phase) for 30 ℃, the centrifugal collection thalline of 5000rpm, 10min, and wash 2 times with quinoline minimal medium (pH 8.0), be resuspended in the quinoline minimal medium, regulate cell density to OD
600=Isosorbide-5-Nitrae ℃ saves backup.
4, to growth and its relation to the degradation effect of quinoline of genus bacillus (Bacillus sp) Q2
Genus bacillus (Bacillus sp) Q2 is inoculated in the minimal medium containing the 500mg/L quinoline, in 30 ℃, the 150r/min shaking table is cultivated, the growth of METHOD FOR CONTINUOUS DETERMINATION cell and the residual concentration of quinoline are as shown in Figure 1, measurement result shows: the growth tendency of genus bacillus (Bacillus sp) Q2 with to the degradation process basic synchronization of quinoline, this bacterium can utilize quinoline to promote self growth as sole carbon, nitrogen and energy substance, and quinoline is had to good degradation effect.
5, the impact of culture condition on the ability of the growth of genus bacillus (Bacillus sp) Q2 and the quinoline of degrading thereof
(1) impact of quinoline starting point concentration: investigate different quinoline starting point concentrations (100, 200, 400, 500, 600, 800, 1000, 1400mg/L) on the impact of genus bacillus (Bacillus sp) Q2 degraded quinoline, in the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, the residual concentration of quinoline as shown in Figure 2, genus bacillus (Bacillus sp) Q2 can be complete by the quinoline-degrading of 500mg/L in 30h, raising along with the quinoline starting point concentration, degradation process obviously extends required lag phase, when quinoline concentration reaches 1400mg/L, genus bacillus (Bacillus sp) Q2 becomes very slow to the removal of quinoline.Result shows, along with the rising of quinoline concentration, quinoline strengthens gradually to the Inhibition of degradation effect of genus bacillus (Bacillus sp) Q2.
(2) impact of culture temperature: investigate the impact of different culture temperature (10,20,30,40 ℃) on genus bacillus (Bacillus sp) Q2 degraded quinoline, in the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, the residual concentration of quinoline as shown in Figure 3, under 30 ℃, through 30h, cultivate, about 500mg/L quinoline is remaining 15.3mg/L only, obviously is better than the removal effect of other temperature.Experimental result shows, the temperature contrast of genus bacillus (Bacillus sp) Q2 degraded quinoline is 30 ℃>40 ℃>20 ℃>10 ℃.
(3) impact of initial pH: investigate the impact of different initial pH value (3,4,5,6,7,8,9,10) on genus bacillus (Bacillus sp) Q2 degraded quinoline, in the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, the residual concentration of quinoline as shown in Figure 4, is determined quinoline-degrading effect under different initial pH value according to the size of quinoline residual concentration in degradation process: pH=9 ≈ pH=10>pH=8>pH=7>pH=6>pH=5>pH=4>pH=3.
(4) impact of inoculum size: investigate the impact of different inoculum size (2vol%, 5vol%, 10vol%, 15vol%, 20vol%, 25vol%) on genus bacillus (Bacillus sp) strain Q2 degraded quinoline, in the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, the residual concentration of quinoline as shown in Figure 5, can find out, inoculum size is larger, quinoline-degrading speed is faster, when inoculum size is 20vol% and 2vol 5%, in shaking flask, the removal speed of quinoline is the highest, show when inoculum size reach 20vol% now more simple dependence increase inoculum size to the impact that improves the quinoline-degrading effect not obvious.
(5) impact of shaking speed: investigate the impact of different shaking speed (50,100,150,200rpm) on genus bacillus (Bacillus sp) Q2 degraded quinoline, in the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, as shown in Figure 6, it is 100,150 and the degradation rate of 200r/min that the quinoline-degrading rate that rotating speed is 50r/min is starkly lower than rotating speed to the residual concentration of quinoline.In addition, after experimental result shows, the three is not fairly obvious on the impact of quinoline-degrading rate, show can meet the demand of genus bacillus (Bacillus sp) Q2 to dissolved oxygen when shaking speed is 100～150r/min, now simple dependence increases speed not remarkable on the impact that improves the quinoline-degrading effect.
Embodiment 2: the intensive treatment of genus bacillus (Bacillus sp.) Q2 to the refinery water of interpolation higher concentration quinoline
In Erlenmeyer flask, genus bacillus (Bacillus sp) Q2 is added in the active sludge of China Petrochemical Industry's Wuhan Company, its removal effect to quinoline of 3 groups of the effects is set.
(a) genus bacillus (Bacillus sp) the Q2 bacteria suspension of 32mL refinery water+33mL quinoline minimal medium+500mg/L quinoline+30mL active sludge+5vol%;
(b) genus bacillus (Bacillus sp) the Q2 bacteria suspension of the active sludge+5vol% after 32mL refinery water+33mL quinoline minimal medium+500mg/L quinoline+30mL autoclaving;
(c) 32mL refinery water+38m quinoline minimal medium+500mg/L quinoline+30mL active sludge.In 30 ℃, the 150rpm shaking table is cultivated.
In the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, the residual concentration of quinoline as shown in Figure 7, result shows: the active sludge that does not add genus bacillus (Bacillus sp) Q2 is slow to the quinoline-degrading in waste water, add genus bacillus (Bacillus sp) Q2 and significantly improved the degradation capability of active sludge to quinoline, and will remove fully containing the quinoline in the refinery water of about 500mg/L quinoline in 32h.
Embodiment 3: the intensive treatment of genus bacillus (Bacillus sp) Q2 to the coking chemical waste water of interpolation higher concentration quinoline
In Erlenmeyer flask, genus bacillus (Bacillus sp) Q2 is added in the active sludge of Wuhan Iron and Steel Plant Coking Company biochemical treatment apparatus, its removal effect to quinoline of 3 groups of the effects is set.
(a) the Q2 bacteria suspension of 32mL coking chemical waste water+33mL quinoline minimal medium+500mg/L quinoline+30mL active sludge+5vol%;
(b) the Q2 bacteria suspension of the active sludge+5vol% after 32mL coking chemical waste water+33mL quinoline minimal medium+500mg/L quinoline+30mL autoclaving;
(c) 32mL coking chemical waste water+38mL quinoline minimal medium+500mg/L quinoline+30mL active sludge.In 30 ℃, the 150rpm shaking table is cultivated.
In the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, as shown in Figure 8, result shows the residual concentration of quinoline: genus bacillus (Bacillus sp) Q2 has played obvious bioaugmentation, and will remove fully containing the quinoline in the coking chemical waste water of about 500mg/L quinoline in 32h.
Embodiment 4: the intensive treatment of genus bacillus (Bacillus sp) Q2 to the tar processing waste water of interpolation higher concentration quinoline
In Erlenmeyer flask, genus bacillus (Bacillus sp) Q2 is added in the active sludge of Wuhan Iron and Steel Plant coking, its removal effect to quinoline of 3 groups of the effects is set.
(a) the Q2 bacteria suspension of 32mL tar processing waste water+33mL quinoline minimal medium+500mg/L quinoline+30mL active sludge+5vol%;
(b) the Q2 bacteria suspension of the active sludge+5vol% after 32mL tar processing waste water+33mL quinoline minimal medium+500mg/L quinoline+30mL autoclaving;
(c) 32mL tar processing waste water+38mL quinoline minimal medium+500mg/L quinoline+30mL active sludge.In 30 ℃, the 150rpm shaking table is cultivated.
In the METHOD FOR CONTINUOUS DETERMINATION nutrient solution, the residual concentration of quinoline as shown in Figure 9, result shows: genus bacillus (Bacillus sp) Q2 has played obvious bioaugmentation, and will remove fully containing the quinoline in the tar processing waste water of about 500mg/L quinoline in 32h.
The genus bacillus of this embodiment (Bacillus sp) Q2 can utilize quinoline to carry out growth and breeding as sole carbon source, nitrogenous source and the energy, in 30h, that the quinoline-degrading of 500mg/L is complete.Genus bacillus (Bacillus sp.) Q2 can tolerate the quinoline of concentration up to 1400mg/L, higher concentration quinoline trade effluent is had to good degradation effect, this bacterium can be applicable to the biological reinforced processing of refinery water, coking chemical waste water and tar processing waste water, has a extensive future.
Claims (3)
1. a strain of quinoline-degrading bacterium, through being accredited as genus bacillus (Bacillus sp), number Q2, be preserved in Chinese Typical Representative culture collection center, preservation date on July 6th, 2011, deposit number is CCTCC NO:M2011239, and the GenBank accession number of bacterial strain 16S rDNA is JN132107, it is characterized in that: genus bacillus (Bacillus sp) Q2 is gram-positive microorganism, bacterium colony is oyster white, and neat in edge is rounded, smooth moistening, central protuberance; This bacterium is hydrogen sulfide production test, V-P test and the nitrate reduction test positive, and indole test, methyl red test and Starch Hydrolysis negative, can utilize quinoline to carry out growth and breeding as sole carbon source, nitrogenous source and the energy.
2. the application of quinoline-degrading bacterium as claimed in claim 1, is characterized in that described genus bacillus (Bacillus sp) Q2 can take quinoline and be grown as sole carbon source and nitrogenous source, for the quinoline of biological degradation trade effluent.
3. the application of quinoline-degrading bacterium as claimed in claim 2, is characterized in that described trade effluent is a kind of in refinery water, coking chemical waste water and tar processing waste water.
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| CN106381315B (en) * | 2016-11-25 | 2019-11-05 | 太原理工大学 | Strengthen the method for quinoline anaerobic degradation and methane phase using the iron filings that get rusty |
| CN114940961B (en) * | 2022-06-21 | 2023-09-15 | 太原理工大学 | Salt-tolerant pyridine degradation strain and application thereof in high-salt pyridine wastewater |
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