CN101255403A - Pyridine degradation bacterium strain and uses thereof - Google Patents
Pyridine degradation bacterium strain and uses thereof Download PDFInfo
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- CN101255403A CN101255403A CNA2007103038710A CN200710303871A CN101255403A CN 101255403 A CN101255403 A CN 101255403A CN A2007103038710 A CNA2007103038710 A CN A2007103038710A CN 200710303871 A CN200710303871 A CN 200710303871A CN 101255403 A CN101255403 A CN 101255403A
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Abstract
The invention discloses a pyridine degradable bacteria and application thereof. The pyridine degradable bacteria are Shinella zoogloeoides BC026 CGMCC No.2224. Applying the inventive Shinella zoogloeoides BC026 in decomposition of pyridine in drain system, not only high decomposition efficiency is obtained, but also the inventive Shinella zoogloeoides BC026 has characteristic of distillage recycle, can exert important function in drain processing, impact endurance of the system is enhanced.
Description
Technical field
The present invention relates to a pyridine degradation bacterium strain and application thereof.
Background technology
Pyridine is a kind of colourless liquid that irritating smell is arranged at normal temperatures, is one of Application and Development kind widest in area in the present heterocyclic arene.Pyridine is poisonous, carcinogenic, teratogenesis, mutagenesis, and easily enrichment in vivo.Can produce harm to human body by suction, picked-up or skin contact, can anaesthetize central nervous system, the eye and the upper respiratory tract are had hormesis.Dizziness, headache, insomnia, instability of gait and tract function disorder appear in long-term the suction, and hepatorenal damage can take place, and cause dermatitis.Pyridine is a kind of good solvent, at industrial denaturing agent, the dyeing auxiliaries of can be used as, and other a series of products, comprises the synthetic initiator of agricultural chemicals, medicine, dyestuff, daily-use chemical industry, spices, fodder additives, rubber ingredients etc.All contain pyridine in a lot of trade effluents, as coking chemical waste water, waste water from dyestuff, pharmacy waste water etc.
Coking chemical waste waters etc. contain the pyridine processing method of industrial waste water generally physico-chemical process, advanced oxidation processes and biological process.Comparatively speaking, the biological process treatment capacity is big, and cost is low, and mild condition does not need special plant and instrument, has the advantage that degraded thoroughly and not produces secondary pollution, is the pollution treatment method of most economical practicality.Yet traditional biologic treating technique is difficult to reach treatment effect, can not satisfy the environmental protection demand.The biological reinforcing technology that occur the seventies in 20th century can improve the scope and the ability of sewage disposal greatly on the basis that does not change existing treatment facility, thereby has been subjected in recent years paying close attention to widely.
Biological reinforcing technology is by add efficient degrading bacteria that screens or the genetic engineering bacterium that obtains through gene recombination from physical environment in Waste Water Treatment, remove certain or certain several pollutent, with the processing power that strengthens Waste Water Treatment, improve processing efficiency.Containing pyridine waste water with the biological einforcement method processing has many advantages, has become the focus of research.
The core of biological reinforcing technology is the microorganism with efficient degradation characteristic that is added.People have isolated a lot of pyridine degradable bacterial strains, wherein major part is a bacterium, as Rhodopseudomonas (Pseudomonas), genus arthrobacter (Arthrobacter), Gordon Salmonella (Gordonia nitida) genus bacillus (Bacillus) etc., actinomycetes and fungi are also arranged, for example have a liking for salt actinomycetes (Nocardioides sp.) and white-rot fungi.These researchs rest on laboratory stage mostly, because a variety of causes such as competition of water quality hydraulics and indigenous bacterium, some biological reinforced system practical application effects are undesirable.Therefore it is very necessary to develop the degradation bacteria strains and the using method thereof that help the sewage disposal practical application.Efficient degrading bacterial strain of the present invention extracts from coking chemical waste water, therefore can better adapt to the wastewater treatment operational mode in actual applications.
In the world seldom, do not see the report of its degraded pyridine at present to the research of Shinella zoogloeoides bacterium.
Summary of the invention
The purpose of this invention is to provide a pyridine degradation bacterium strain and application thereof.
Pyridine degradable bacteria provided by the present invention is Shinella zoogloeoides BC026 CGMCC № .2224.
Described Shinella zoogloeoides BC026 CGMCC № .2224 derives from Shoudu Iron and Steel Co coke-oven plant Sewage treatment systems second pond returned sluge, obtains through domestication, separation, purifying.This bacterial strain is negative gram bacillus, and is aerobic, and mobility is arranged, and is tawny, ganoid circular small colonies in solid medium, and the autoflocculation phenomenon is arranged in the liquid medium within.Shinella zoogloeoides BC026 CGMCC № .2224 has resistance to penbritin, sulphuric acid kanamycin, spectinomycin, to paraxin, tsiklomitsin, cephalo sensitivity.Well-grown in Ah Xu shellfish nitrogen-free agar carries two big plasmids.The 16SrRNA sequence of Shinella zoogloeoides BC026 CGMCC № .2224 is shown in sequence in the sequence table 1.
Described Shinella zoogloeoides BC026 CGMCC № .2224, be preserved in Chinese microorganism strain preservation board of trustee reason person on October 23rd, 2007 and understand common micro-organisms center (abbreviation CGMCC, the address is: No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north), preserving number is CGMCC № .2224.
Shinella zoogloeoides BC026 of the present invention can be used for the degraded of pyridine in the sewage system, concrete grammar can be Shinella zoogloeoides BC026 CGMCC № .2224 is suspended in the sewage that contains pyridine and/or its derivative, at 20-35 ℃, the pH value is cultivated under the condition of 5-9.
The temperature that described Shinella zoogloeoides BC026 CGMCC № .2224 cultivates in sewage is preferably 30-35 ℃, and the best is 30 ℃, and pH value condition optimization is pH7-pH8, and the best is pH8.
The bacteria agent that is activeconstituents with above-mentioned Shinella zoogloeoides BC026 also belongs to protection scope of the present invention; Can add acceptable auxiliary as required in this microbial inoculum.
Shinella zoogloeoides BC026 of the present invention, when throwing the bacterium amount for 0.1g/L, at 30 ℃, 180r/min, the pH value is under 7 the condition, the BC026 bacterium can be in 17h be that the pyridine of 400mg/L is degraded fully with concentration.At the pyridine starting point concentration is in the minimal medium of 99～1806mg/L, and the BC026 bacterium all can keep higher degrading activity; The degraded optimum temperuture is 30 ℃, and optimum pH is 8.Inferred that by the nitrogen transformation experiment the first step of BC026 bacterium degraded pyridine is to disconnect two C-N chains, generates ammonia nitrogen and glutaraldehyde, glutaraldehyde is oxidized to pentanedioic acid subsequently, and finally is converted into carbonic acid gas and water.Pyridine has 50% nitrogen transformation to become ammonia nitrogen approximately in the whole degradation process.
Utilize Shinella zoogloeoides BC026 of the present invention to be used for the degraded of sewage system pyridine, not only has very high degradation efficiency, Shinella zoogloeoides BC026 of the present invention is the characteristic with autoflocculation simultaneously, can play an important role the shock-resistance of enhanced system in the sewage disposal.
Shinella zoogloeoides BC026 of the present invention is taken from treatment unit for waste water, is the dominant bacteria in the Waste Water Treatment, can adapt to complicated physical condition, thereby the having a extensive future of this bacterial strain.Shinellazoogloeoides BC026 of the present invention carries two big plasmids, and the pyridine degradable gene of may encoding is for the structure of later genetic engineering bacterium has been created condition.
Description of drawings
Fig. 1 is the BC026 stereoscan photograph.
Fig. 2 is the biological degradation of BC026 bacterium to pyridine.
Fig. 3 is the degraded situation of BC026 bacterium to the different concns pyridine.
Fig. 4 is the degradation characteristic research of pyridine under the differing temps.
Fig. 5 be under the different pH BC026 bacterium to the biodegradation character of pyridine.
Fig. 6 is the change in concentration of pyridine and ammonia nitrogen in the BC026 bacterium degradation process.
Fig. 7 is the possible pathways metabolism of BC026 bacterium to pyridine.
Fig. 8 is a BC026 plasmid electrophorogram.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.
Separation, purifying and the evaluation thereof of embodiment 1, Shinella zoogloeoides BC026
1, the separation of Shinella zoogloeoides BC026 and purifying
Shinella zoogloeoides BC026 takes a sample from the active sludge of Shoudu Iron and Steel Co coke-oven plant Sewage treatment systems second pond, the strain Gram-negative bacteria that process domestication, separation and purifying obtain.Concrete steps are as follows:
Get Shoudu Iron and Steel Co Coking Plant Wastewater treatment system second pond returned sluge.In the container that the mud sample is housed, drip a small amount of pyridine solution (making pyridine concentration keep 100-200mg/L), intermittent aeration, regularly add 10mL pyridine (300mg/L) and the former coking chemical waste water of 50mL, (every L contains 8.5g KH to add the 2mL phosphate buffered saline buffer simultaneously
2PO
4, 21.75gK
2HPO
4, 33.4gNa
2HPO
47H
2O, 1.7gNH
4Cl, pH are 7.2) and the 2mL trace element solution (every L contains 1.5g FeCl
36H
2O, 0.024gNiCl
26H
2O, 0.19g CoCl
26H
2O, 0.002gCuCl
22H
2O, 0.024g Na
2MoO
42H
2O, 0.07g ZnCl
2, 0.006g H
3BO
3), tamed 60 days.
Get nutrient solution that the above-mentioned domestication of 200mL obtains in triangular flask, add sodium pyrophosphate solution and several glass microballons of 2 0.01%, at 30 ℃, concussion 30min fully shakes up in the shaking table of 180r/min, and is centrifugal then.Precipitation is transferred to the minimal medium that 100mL contains the 500mg/L pyridine (to be contained among every L: Na
2HPO
412H
2O 1.42g, KH
2PO
41.36g, MgSO
47H
2O 0.216g, CaCl
20.006g, MnSO
4H
2O 1.69g, CoCl
26H
2O0.00024g, H
3BO
30.00116g, Na
2MoO
42H
2O 0.000024g, FeSO
47H
2O 0.00278g, ZnSO
47H
2O 0.00115g, CuSO
45H
2O 0.00038g) cultivate in, 30 ℃, shaking table 180r/min cultivates, after about 4 days, get 5mL bacterium liquid in minimal medium under the same terms switching cultivate twice.At this moment, with the sterilization deionized water bacterium liquid is diluted 10
-1To 10
-7Doubly, respectively get the coating in inorganic salt solid medium (add 2% agar in the above-mentioned inorganic salt liquid substratum and make) flat board of 0.1mL diluent and separate, 30 ℃ of cultivations, grow obvious bacterium colony after, the representational single bacterium colony of picking, line separation and purification.Obtain the bacterial strain that 7 strains can be grown on the inorganic salt solid medium, it is in 15% the glycerine that the LB nutrient solution of these bacterial strains is kept at final concentration in-70 ℃, gives numbering respectively.
By the growth velocity and the detection pyridine degradable situation of observing bacterium, the ability of determining a strain bacterial strain (BC026) degraded pyridine is the strongest, this strain bacterium has the characteristic (naked eyes can be observed and occur cotton-shaped cenobium in the nutrient solution after the strain culturing) of autoflocculation in liquid culture simultaneously, this specific character will help it to form zoogloea in Sewage treatment systems, strengthen the shock-resistance of biological treatment system, thereby this bacterium has higher researching value.
BC026 liquid culture condition is: add the 100mL substratum in the 250mL Erlenmeyer flask, as unique carbon, nitrogenous source, shaking table is made as 30 ℃, 180r/min with the 500mg/L pyridine.Add the 500mg/L pyridine in the LB substratum, keep the degradation by bacteria vigor, prevent assorted bacterium intrusion.By centrifugal collection thalline, centrifugal condition is 4000r/min, 10min.
This BC026 bacterial strain is negative gram bacillus, and is aerobic, and mobility is arranged, and is tawny, ganoid circular small colonies in solid medium, and the autoflocculation phenomenon is arranged in the liquid medium within.This bacterium has resistance to penbritin, sulphuric acid kanamycin, spectinomycin, to paraxin, tsiklomitsin, cephalo sensitivity.Well-grown in Ah Xu shellfish nitrogen-free agar.The stereoscan photograph of BC026 as shown in Figure 1.
This BC026 bacterial strain is delivered the physiological and biochemical property evaluation that Institute of Micro-biology carries out, and qualification result is as shown in table 1.
The physiological and biochemical property of table 1.BC026 bacterial strain
| Experimental project | The result | Experimental project | The result | Experimental project | The result | Experimental project | The result |
| Cellular form | Shaft-like | Gramstaining | Negative | ||||
| Contrast | - | The D-melibiose | - | The p-phenylglycollic acid | - | The L-Histidine | + |
| The a-cyclodextrin | - | Methyl glucoside | - | Methylene-succinic acid | - | Oxyproline | + |
| Dextrin | + | The D-psicose | + | A-chloro butyric acid | + | The L-leucine | - |
| Glycogen | + | The D-raffinose | - | A-chloro pentanedioic acid | - | The L-ornithine | + |
| Tween40 | - | The D-rhamnosyl | + | A-chloro valeric acid | - | The L-phenylalanine | - |
| Tween80 | + | The D-sorbyl alcohol | + | D, L-lactic acid | + | The L-proline(Pro) | + |
| N-acetylgalactosamine | - | Sucrose | + | Propanedioic acid | - | The L-Pyrrolidonecarboxylic acid | + |
| The N-acetylglucosamine | + | The D-trehalose | + | Propionic acid | + | The D-Serine | - |
| Ribitol | + | Turanose | + | Quinic acid | - | The L-Serine | + |
| L-arabinose | + | Xylitol | - | The D-saccharinic acid | - | The L-Threonine | + |
| The D-arabitol | + | Pyruvic Acid Methyl ester | + | The certain herbaceous plants with big flowers diacid | - | D, the L-carnitine | + |
| The D-cellobiose | + | Succinic Acid-methyl esters | + | Succinic Acid | + | The g-aminobutyric acid | + |
| The i-tetrahydroxybutane | + | Acetate | + | Bromosuccinic acid | + | Urocanic acid | + |
| D-fructose | + | Suitable-aconic acid | - | Succinamic acid | + | Inosine | + |
| L-fructose | + | Citric acid | - | Glucuronamide | - | Uridine | + |
| The D-semi-lactosi | + | Formic acid | + | The L-alanimamides | + | Thymidine | - |
| Gentiobiose | - | The D-galactonolactone | - | The D-L-Ala | + | Phenyl-ethyl amine | - |
| A-D-glucose | + | The D-galacturonic acid | - | The L-L-Ala | + | Putrescine | - |
| The m-inositol | + | The D-glyconic acid | - | The L-alanyl-glycine | + | The 2-monoethanolamine | + |
| The a-D-lactose | - | The b-methyl glucoside | - | Altheine | + | 2, the 3-butyleneglycol | - |
| Lactulose | - | The D-glucuronic acid | - | The L-aspartic acid | + | Glycerine | + |
| Maltose | + | The a-hydroxybutyric acid | + | L-L-glutamic acid | + | D, the L-glycerophosphate | + |
| D-N.F,USP MANNITOL | + | The b-acetaldol | + | Glycosides aminoacyl aspartic acid | + | D-glucose-1-phosphoric acid | - |
| The D-seminose | + | The g-hydroxybutyric acid | - | Glycosides aminoacyl L-glutamic acid | b | D-glucose-6-phosphoric acid | - |
2, the 16S rDNA of Shinella zoogloeoides BC026 identifies
Identify by 16S rRNA sequential analysis and Biolog microbial identification system, determine that the BC026 bacterium is Shinella zoogloeoides.Concrete steps are as follows:
Extract the genomic dna of BC026 bacterium with the TIANGEN genome DNA extracting reagent kit, as the template of PCR reaction, the design primer carries out the amplification of PCR segment genome.The upstream and downstream primer sequence is respectively:
27F：5’AGAGTTTGATCATGGCTCAG 3’
1492R：5’TACGGTTACCTTGTTACGACTT 3’
The PCR setting program is: 94 ℃ of pre-sex change 2min of elder generation; 94 ℃ of sex change 1min then, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 30 circulations; Last 72 ℃ are extended 15min, 4 ℃ of preservations eventually.The PCR product separates with 0.8% sepharose, purifying and the order-checking of recovery back.The nucleotide sequence that the 16S rDNA of sequencing result BC026 bacterium has sequence 1 in the sequence table.
The 16S rRNA sequencing result of BC026 imported carry out the homology comparison among the GenBank, the result shows that the homology of it and Crabtreella saccharophila (AB238789), Zoogloea ramigera (X74915), Mycoplana sp. (AB248361) is 99%.In order to confirm breadboard qualification result, entrust Institute of Microorganism, Academia Sinica to carry out specialty and identify, according to the multinomial physio-biochemical characteristics of this bacterium, and the analysis of Biolog microbial identification system, determine that BC026 belongs to Shinella zoogloeoides.
Shinella zoogloeoides different name of the same race is Crabtreella saccharophila and gives birth to branch moving glue bacterium (Zoogloea ramigera), reclassifies back called after Shinella zoogloeoides, and does not have Chinese translation temporarily in 2006.
Therefore, this bacterium is Shinella zoogloeoides BC026 by definite designation, be preserved in Chinese microorganism strain preservation board of trustee reason person on October 23rd, 2007 and understand common micro-organisms center (abbreviation CGMCC, the address is: No. 13, one in Zhong Guan-cun, Haidian District, BeiJing, China city north), preserving number is CGMCC № .2224.
3, Shinella zoogloeoides BC026 is an azotobacter
According to document, giving birth to the moving glue bacterium of branch is azotobacter.Cultivate by Ah Xu shellfish nitrogen-free agar, prove that the BC026 bacterium is a strain azotobacter.Concrete grammar is:
The BC026 bacterium is coated on Ah Xu the shellfish nitrogen-free agar, and Ah Xu shellfish nitrogen-free agar consists of C
7H
12O
6, 10g/L; KH
2PO
4, 0.2g/L; MgSO
47H
2O, 0.2g/L; NaCl, 0.2g/L; CaSO
42H
2O, 0.1g/L; CaCO
3, 5g/L.Observing the BC026 bacterium can well grow on substratum, judges that with this BC026 bacterium is a strain azotobacter.
1, Shinella zoogloeoides BC026 CGMCC № .2224 detects the biodegradability of pyridine
Be under the condition of sole carbon, nitrogenous source with the pyridine, investigating the degradation capability of BC026 pyridine.Find that the BC026 bacterium has removal effect preferably to pyridine, can finish degraded within a short period of time.Concrete steps are as follows:
1) gets the LB nutrient solution that 2mL is cultured to the Shinella zoogloeoides BC026 CGMCC № .2224 of logarithmic phase, collect bacterial sediment, be inoculated in the triangular flask that the 200mL minimal medium is housed, be cultured to logarithmic phase.
2) centrifugal bacterium liquid, gained Shinella zoogloeoides BC026 bacterial sediment resuspended, centrifugal three the impurity of minimal medium with removal Shinella zoogloeoides BC026 bacterium surface adsorption, transfer to again in the sterilization inorganic salt liquid substratum of certain volume, be prepared into bacterium liquid optical density value OD
602Be 1～2 Shinellazoogloeoides BC026 bacteria suspension,, measure actual inoculum size with the dry weight method simultaneously as the inoculation liquid of degradation experiment.
3) with step 2) the Shinella zoogloeoides BC026 bacteria suspension of preparation is that 0.1g/L (dry weight method mensuration) is inoculated in 100mL and contains in the minimal medium of 400mg/L pyridine with the dry mycelium inoculum size, with sealing film phonograph seal, at 30 ℃, shaking culture in the shaking table of 180rpm.If do not add the minimal medium that contains the 400mg/L pyridine of bacterium, carry out experiment under the equal conditions as blank.
4) in the described culturing process of step 3), be taken at the reaction solution of described processing of step 3) and blank at interval in 1～2 hour respectively, measure pyridine concentration and bacterium liquid absorbancy.Detection method is as described below:
Nectar degree:, be that bacterium liquid absorbancy is detected at the 602nm place in absorbing wavelength with Tianjin, island UV2401 type ultraviolet-visible spectrophotometer.
Pyridine concentration: fermentation broth sample by 0.22 μ m membrane filtration after, by high performance liquid chromatography (Tianjin, island LC10ADVP, SPD10AVP UV-Vis Detector; Diamonsil C18 chromatographic column, 250 * 4.6mm, 5 μ m) mensuration pyridine concentration.
The result as seen from Figure 2, does not inoculate in the blank assay of bacterium liquid as shown in Figure 2, and the pyridine change in concentration is less, reduces due to a small amount of volatilization that partly should be pyridine; And in the degradation experiment of inoculation bacterium liquid, along with the reduction of pyridine concentration, the nectar degree increases, and after pyridine consumption fully, the nectar degree is decayed fast.This explanation BC026 bacterium can pyridine be sole carbon, nitrogenous source, but and within a short period of time pyridine is degraded fully.After the BC026 bacterium was inoculated into nutrient solution, the absorbed portion pyridine made pyridine concentration reduce rapidly at the 4h of beginning immediately.In 6h, nectar degree rate of rise begins to accelerate at 4h, and this explanation BC026 bacterium begins the pyridine of degrading through having adapted to growing environment inhibition period.After the 6h, bacterium reaches logarithmic phase, and pyridine degradable speeds up.At 15.5h, the pyridine clearance reaches 98.7%, and this moment, the pyridine as carbon, nitrogenous source lacked, and bacterium is dead because of can not get enough nutrition, so the nectar degree descends rapidly.A is the pyridine concentration curve of blank among Fig. 2; B is BC026 bacterium pyridine concentration curve in the culturing process in the minimal medium that contains the 400mg/L pyridine among Fig. 2; C is BC026 bacterium nectar degree (OD602) change curve in the culturing process in the minimal medium that contains the 400mg/L pyridine among Fig. 2.
2, the degradation characteristic of Shinella zoogloeoides BC026 bacterium under different pyridine starting point concentrations
By the increase of the discovery of the degradation experiment under the different pyridine starting point concentrations along with the pyridine starting point concentration, degraded is elongated inhibition period, and the pyridine degradable speed of degradative phase accelerates.Concrete steps are as follows:
1) gets the LB nutrient solution that 2mL is cultured to the Shinella zoogloeoides BC026 CGMCC № .2224 of logarithmic phase, collect bacterial sediment and be inoculated in the triangular flask that the 200mL minimal medium is housed, be cultured to logarithmic phase.
2) centrifugal bacterium liquid, gained Shinella zoogloeoides BC026 bacterial sediment resuspended, centrifugal three the impurity of minimal medium with removal Shinella zoogloeoides BC026 bacterium surface adsorption, transfer to again in the sterilization inorganic salt liquid substratum of certain volume, be prepared into bacterium liquid optical density value OD
602Be 1～2 Shinellazoogloeoides BC026 bacteria suspension, as the inoculation liquid of degradation experiment.Measure actual inoculum size with the dry weight method simultaneously.
3) in 4 250mL triangular flasks that the 100mL minimal medium is housed, add pyridine respectively, its final concentration is respectively 99mg/L, 473mg/L, 932mg/L, 1806mg/L, then respectively with step 2) the Shinella zoogloeoides BC026 bacteria suspension for preparing is that 0.06g/L is inoculated in the above-mentioned triangular flask with the dry mycelium inoculum size, with inoculation not 100mL being housed, to contain the pyridine final concentration be that the 250mL triangular flask of the minimal medium of 1896mg/L is a blank, with each triangular flask with sealing film phonograph seal, at 30 ℃, shaking culture in the shaking table of 180rpm.
4) in the described culturing process of step 3), be taken at the reaction solution of described processing of step 3) and blank at interval in 1～2 hour respectively, measure pyridine concentration and bacterium liquid absorbancy.Pyridine concentration and bacterium liquid absorbency detection method are as described in the step 4) in the step 1.
The result as shown in Figure 3, wherein the pyridine concentration curve is shown in B among Fig. 3, by B among Fig. 3 is in the nutrient solution of 99～1806mg/L at the pyridine starting point concentration as can be seen, Shinella zoogloeoides BC026 bacterium is the degradable pyridine all, but under each pyridine starting point concentration condition, the length of inhibition period is different with degradation rate.At the pyridine starting point concentration is in the nutrient solution of 99mg/L, and Shinella zoogloeoides BC026 does not almost show inhibition period, and pyridine concentration straight line descends.Along with the rising of pyridine starting point concentration, time inhibition period is elongated, illustrates that the pyridine starting point concentration is high more, and bacterium adapts to slow more.But in case enter degradative phase, microbiological deterioration speed is fast in the high nutrient solution of pyridine starting point concentration, see that from figure to be slope absolute value bigger, this is because bacterium has adapted to growing environment fully, the pyridine of high density provides more sufficient nutrient matrix to be engaged in vital movement and a large amount of breeding to bacterium, and after bacterial number increased, required carbon, nitrogenous source were many, the corresponding raising of the degradation rate of pyridine, the two is mutually promoted.
The change curve of bacterium liquid absorbancy is shown in A among Fig. 3; Among Fig. 3 A as can be seen, under four concentration, bacterium amount all raises along with the degraded of pyridine.At the pyridine starting point concentration is in the nutrient solution of 99mg/L, the nectar line of writing music peaks very soon, with B contrast among Fig. 3, show at the pyridine starting point concentration to be that the pyridine concentration of the nutrient solution of 99mg/L is when dropping to 20mg/L, the very few limiting factor that becomes Shinella zoogloeoides BC026 growth of available matrix, Shinella zoogloeoides BC026 bacterium amount begins to descend.From the pyridine starting point concentration be 1806mg/L bacterium liquid absorbancy change curve as can be seen: experiment initial stage bacterial growth speed is slower, the degradation rate of pyridine is also slow, but along with the degraded of pyridine, high-purity pyridine weakens gradually to the restraining effect of Shinella zoogloeoides BC026 growth; When pyridine concentration was reduced to the 400mg/L left and right sides, Shinella zoogloeoides BC026 grew into logarithmic phase, and the bacterium amount increases rapidly.Under different pyridine concentration, the maximum nectar degree and the required time that can reach have nothing in common with each other, article four, in the curve, maximum nectar degree is respectively: 0.217g/L, 0.623g/L, 0.857g/L, 1.375g/L, required time is respectively: 15.5h, 17.5h, 27.5h and 56.5h.Along with the rising of pyridine starting point concentration, in the nutrient solution quantitative change of carbon, nitrogenous source big, maximum nectar degree increases progressively.But this growth is not directly proportional, this explanation, and the limiting factor of the growth of Shinella zoogloeoides BC026 not only has the concentration of pyridine, also has other factor, and is for example relevant to the competition of living space with the accumulation and the bacterium of meta-bolites.99mg/L among Fig. 3 among the A, 473mg/L, 932mg/L, 1806mg/L refer to that respectively Shinella zoogloeoides BC026 contains in the substratum that pyridine concentration is 99mg/L, 473mg/L, 932mg/L or 1806mg/L the change curve of nectar degree (OD602) in the culturing process above-mentioned; 99mg/L among Fig. 3 among the B, 473mg/L, 932mg/L, 1806mg/L, blank refer to that respectively Shinella zoogloeoides BC026 contains pyridine concentration curve in culture medium culturing that pyridine concentration is 99mg/L, 473mg/L, 932mg/L or 1806mg/L or the above-mentioned blank treating processes above-mentioned.
3, the degradation characteristic of Shinella zoogloeoides BC026 bacterium under differing temps
Under differing temps, implement degradation experiment, find that the BC026 bacterium has higher pyridine degradable efficient at 30～35 ℃, from angle of practical application, select 30 ℃ comparatively suitable.Concrete steps are as follows:
After the adding final concentration is the 500mg/L pyridine in minimal medium, with the step 2 in the step 2) the Shinella zoogloeoides BC026 bacteria suspension for preparing is that 0.14g/L is inoculated in this substratum with the dry mycelium inoculum size, branch is got 100mL nutrient solution branch and is filled in four 250mL triangular flasks, places 20 ℃, 25 ℃, 30 ℃, 35 ℃ shaking table to cultivate respectively.With one 100mL being housed, not connect bacterium, final concentration be that the minimal medium of 500mg/L pyridine is cultivated in 35 ℃ shaking table simultaneously, to be made as blank.Take a sample according to the described method of the step 4) in the step 1 then, measure the pyridine change in concentration in the culturing process, draw the pyridine concentration curve.
The result as shown in Figure 4, the result shows, throw bacterium amount (0.14g/L) because experiment has strengthened dry mycelium, so bacterium directly enters the logarithm degradative phase.As can be seen from Figure 4, the degradation speed in the time of 20 ℃ is the slowest, and the speed in the time of 25 ℃ is fast slightly, and the degradation rate when 30 ℃ and 35 ℃ is the highest.30 ℃ of degradation capabilities with 35 ℃ differ very little, and the degradation rate when reaction 12h almost reaches 85% simultaneously.The suitableeest growth, the degradation temperature of this explanation bacterium may be between 30～35 ℃, considers the practical situation of sewage treatment process, and 30 ℃ of the culture temperature selections of BC026 bacterium are comparatively reasonable.20 ℃, 25 ℃, 30 ℃, 35 ℃ of marks among Fig. 4, blank to be respectively above-mentioned Shinellazoogloeoides BC026 cultivate under 20 ℃, 25 ℃, 30 ℃ or 35 ℃ of conditions or above-mentioned blank treating processes in the pyridine concentration curve.
4, the degradation characteristic of Shinella zoogloeoides BC026 bacterium under different pH values
By the degradation experiment of the initial pH value of difference condition, find that the suitableeest degraded pH value of BC026 bacterium is 8.Concrete steps are as follows:
The minimal medium of the equal volume of in 7 250mL triangular flasks, packing into, regulate pH to 4,5,6,7,8,9,10 respectively, all be the step 2 in the 0.13g/L inoculation step 2 with the inoculum size) the Shinellazoogloeoides BC026 bacteria suspension for preparing, and to add pyridine to final concentration respectively be 500mg/L, and the nutrient solution final volume is 100mL.With each triangular flask with sealing film phonograph seal, at 30 ℃, shaking culture in the shaking table of 180rpm.In the culturing process, take a sample, measure the pyridine change in concentration in the culturing process, draw the pyridine concentration curve according to the described method of the step 4) in the step 1.
The result draws from Fig. 5 analysis as shown in Figure 5, and the pH value is 4 and 10 o'clock, and the BC026 bacterium does not have Degradation to pyridine.PH is that solution is very limpid in 4 the nutrient solution, and cell condensation becomes the small-particle precipitation.The pH value is 10 o'clock, and the part composition in the substratum generates precipitation, has destroyed the substratum composition.Be determined at the pH value in the experiment and be that the bacterium amount slightly reduces under 4 and 10 the condition.Be that bacterium can both be unique carbon, nitrogenous source growth with pyridine under 5～9 the condition at pH, the speed difference of degraded just, the fastest pH value of degrading is 8, other is followed successively by pH7＞pH6 ≈ pH5＞pH9.PH=4 among Fig. 5, pH=5, pH=6, pH=7, pH=8, pH=9, pH=10 refer to that respectively Shinella zoogloeoides BC026 is a pyridine concentration curve in the culturing process under 4,5,6,7,8,9 or 10 conditions at pH.
5, Shinella zoogloeoides BC026 bacterium is to the detection of pyridine pathways metabolism
The ammonia nitrogen concentration in the mensuration degradation process and the variation relation of pyridine are inferred the possible pathways metabolism of BC026 bacterium to pyridine.Concrete steps are as follows:
1) gets the LB nutrient solution that 2mL is cultured to the Shinella zoogloeoides BC026 CGMCC № .2224 of logarithmic phase, collect bacterial sediment and be inoculated in the triangular flask that the 200mL minimal medium is housed, be cultured to logarithmic phase.
2) centrifugal bacterium liquid, gained Shinella zoogloeoides BC026 bacterial sediment resuspended, centrifugal three the impurity of minimal medium with removal Shinella zoogloeoides BC026 bacterium surface adsorption, transfer to again in the sterilization inorganic salt liquid substratum of certain volume, be prepared into bacterium liquid optical density value OD
602Be 1～2 Shinellazoogloeoides BC026 bacteria suspension, as the inoculation liquid of degradation experiment.Measure actual inoculum size with the dry weight method simultaneously.
3) with step 2) the Shinella zoogloeoides BC026 bacteria suspension for preparing is that 0.1g/L is inoculated in 100mL and contains in the minimal medium of 400mg/L pyridine with the dry mycelium inoculum size, with sealing film phonograph seal, at 30 ℃, shaking culture in the shaking table of 180rpm.Get the minimal medium that does not add bacterium, contains the 400mg/L pyridine and carry out cultivation under the same terms, to be made as blank.
4) 2～3h gets and cultivates bacterium liquid sample at interval, according to the concentration of following step measurements pyridine and ammonia nitrogen.3～4h gets and cultivates bacterium liquid sample at interval, according to following step measurements intermediate product.
Detection method is:
Pyridine concentration: sample by 0.22 μ m membrane filtration after, by high performance liquid chromatography (Tianjin, island LC10ADVP, SPD10AVP UV-Vis Detector; Diamonsil C18 chromatographic column, 250mm * 4.6mm, 5 μ m) measure.
Ammonia nitrogen concentration: sample is measured with Whitfield's ointment-hypochlorite light-intensity method (GB 7481-87) through 0.22 μ m membrane filtration.
Intermediate product: sample is through 0.22 μ m membrane filtration, the extraction of methylene dichloride equal-volume, filter with the anhydrous sodium sulphate of oven dry in advance then, go up qualitative analysis at GC/MS (Agilent 6890N GC/5973MSD, DB-5MS capillary column, 30m * 0.25mm * 0.25 μ m).
The result is shown by Fig. 6 as shown in Figure 6, produces ammonia nitrogen in the degradation process.When pyridine began to reduce, the amount of ammonia nitrogen raise, and the first step of this explanation pyridine degradable is just sloughed nitrogen-atoms, and nitrogen molecule is discharged in the solution with the form of ammonia nitrogen.After pyridine was degraded fully, the amount of ammonia nitrogen no longer increased, and this further proves does not have other intermediate product denitrogenation to be transformed into ammonia nitrogen.And can impel and glutaraldehyde, the relevant coenzyme generation of glutatate dehydrogenase according to the document pyridine, infer in conjunction with the complete symmetrical structure of pyridine, opened two C-N keys during the pyridine denitrogenation.After pyridine degradable was complete, the amount of ammonia nitrogen began to reduce, and can serve as the material of carbon source in this proof nutrient solution in addition, i.e. mesostate after the pyridine denitrogenation.Sampling is measured by GC-MS in the experimentation, does not detect mesostate, and this is identical with bibliographical information.PH value in the degradation process reduces along with the degraded of pyridine, and after pyridine was degraded fully, the pH value began to raise.This explanation pyridine degradable produces the tart intermediate product, according to bibliographical information, infer that this mesostate might be a pentanedioic acid, and this material may be by the glutaraldehyde oxidation.Glutaraldehyde may not be the product oxidation via pyridine denitrogenation hydroxylation gained, so generates glutaraldehyde after inferring the pyridine denitrogenation, and this is that Padil is different with propanedioic acid with the pyridine intermediate product of bibliographical information.About 17h, the ammonia nitrogen amount no longer reduces, and the ammonia nitrogen available a kind of nitrogenous source that is the BC026 bacterium, according to bibliographical information, giving birth to the moving glue bacterium of branch is chemoheterotrophic bacteria, lack available organic carbon in this explanation culture system, so the deducibility pentanedioic acid further is converted into the unserviceable inorganic carbon of BC026.Generally speaking, biology is converted into a part of carbon outside the living matter of self by the assimilation and dissimilation effect, and other carbon is by respiration, and finally the form with carbonic acid gas discharges, and therefore this inorganic carbon may be carbonic acid gas.By the corresponding relation of pyridine and ammonia nitrogen as can be seen, in degradation process, the nitrogen in the pyridine has 50% to be converted into ammonia nitrogen approximately.Other has 50% nitrogen to exist with other forms in vivo or in the environment.Pyridine-N among Fig. 6 is a pyridine concentration curve in the BC026 culturing process, and ammonia nitrogen is a nitrogen concentration change curve in the BC026 culturing process.
In sum, the pathways metabolism of supposition when the BC026 bacterium is sole carbon, nitrogenous source with the pyridine as shown in Figure 7.Continuation is cultivated bacterium liquid, the amount of ammonia nitrogen does not have to change substantially, after in nutrient solution, adding the sterilization glucose solution, ammonia nitrogen amount in the mensuration system, find that ammonia nitrogen concentration is reduced to zero fast, this is because pyridine is sole carbon, a nitrogenous source in the nutrient solution, and therefore the carbon of pyridine, nitrogen prove that than best carbon, nitrogen ratio much smaller than microorganism growth the bacterium decline that late stage of culture shows causes by lacking carbon source.
6, the plasmid of Shinella zoogloeoides BC026 bacterium extracts
The method that provides with the QIAGEN test kit is extracted the plasmid in the BC026 bacterium.Having proved has two big plasmids among the Shinellazoogloeoides BC026, its function is also in further verifying.Concrete steps are as follows:
1) adding final concentration in the LB nutrient solution is the pyridine of 500mg/L, and inoculation Shinella zoogloeoidesBC026 is cultured to the logarithm later stage.
2) extract plasmid according to the method for qiagen plasmid extraction test kit.
3) separate with agarose gel electrophoresis.
The result as shown in Figure 8, the result shows that two big plasmids (plasmid 1 band among Fig. 8, plasmid 2 bands) are arranged among the Shinella zoogloeoides BC026.Among Fig. 8, swimming lane L1 is a molecular weight standard, and swimming lane L2-L7 is a Shinella zoogloeoides BC026 gel electrophoresis separating spectrum.
Sequence table
<160>1
<210>1
<211>1449
<212>DNA
<213>Shinella zoogloeoides
<400>1
agagtttgat catggctcag aacgaacgct ggcggcaggc ttaacacatg caagtcgaac 60
gccccgcaag gggagtggca gacgggtgag taacgcgtgg gaatctaccc atctctacgg 120
aataactcag ggaaacttgt gctaataccg tatacgccct tcgggggaaa gatttatcgg 180
agatggatga gcccgcgttg gattagctag ttggtggggt aaaggcctac caaggcgacg 240
atccatagct ggtctgagag gatgatcagc cacattggga ctgagacacg gcccaaactc 300
ctacgggagg cagcagtggg gaatattgga caatgggcgc aagcctgatc cagccatgcc 360
gcgtgagtga tgaaggccct agggttgtaa agctctttca ccggtgaaga taatgacggt 420
aaccggagaa gaagccccgg ctaacttcgt gccagcagcc gcggtaatac gaagggggct 480
agcgttgttc ggaattactg ggcgtaaagc gcacgtaggc gggtatttaa gtcaggggtg 540
aaatcccgga gctcaactcc ggaactgcct ttgatactgg gtacctagag tatggaagag 600
gtaagtggaa ttccgagtgt agaggtgaaa ttcgtagata ttcggaggaa caccagtggc 660
gaaggcggct tactggtcca ttactgacgc tgaggtgcga aagcgtgggg agcaaacagg 720
attagatacc ctggtagtcc acgccgtaaa cgatgaatgt tagccgtcgg catgcatgca 780
tgtcggtggc gcagctaacg cattaaacat tccgcctggg gagtacggtc gcaagattaa 840
aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgaagc 900
aacgcgcaga accttaccag cccttgacat gtcggtcgcg gattacagag atgttttcct 960
tcagttaggc tggaccgaac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat 1020
gttgggttaa gtcccgcaac gagcgcaacc ctcgccctta gttgccagca ttcagttggg 1080
cactctaagg ggactgccgg tgataagccg agaggaaggt ggggatgacg tcaagtcctc 1140
acggccctta cgggctgggc tacacacgtg ctacaatggt ggtgacagtg ggcagcgaga 1200
cagcgatgtc gagctaatct ccaaaagcca tctcagttcg gattgcactc tgcaactcga 1260
gtgcatgaag ttggaatcgc tagtaatcgc ggatcagcat gccgcggtga atacgttccc 1320
gggccttgta cacaccgccc gtcacaccat gggagttggt tttacccgaa ggcgatgcgc 1380
taaccgcaag gaggcagtcg accacggtag ggtcagcgac tggggtgaag tcgtaacaag 1440
gtaaccgta 1449
Claims (7)
1、Shinella zoogloeoides BC026 CGMCC №.2224。
2, the application of Shinella zoogloeoides BC026 CGMCC № .2224 in nitrogenous heterocyclic compound degradation.
3, application according to claim 2 is characterized in that: described nitrogen-containing heterocycle compound is the pyridine or derivatives thereof.
4, application according to claim 3, it is characterized in that: described nitrogenous heterocyclic compound degradation is pyridine and/or its derivative in the degradation of sewage, concrete grammar is for to be suspended in Shinella zoogloeoides BC026 CGMCC № .2224 in the sewage that contains pyridine and/or its derivative, at 20-35 ℃, the pH value is cultivated under the condition of 5-9.
5, application according to claim 4 is characterized in that: the temperature that described Shinella zoogloeoides BC026CGMCC № .2224 cultivates in sewage is 30-35 ℃, and pH value condition is pH7-pH8.
6, application according to claim 5 is characterized in that: the temperature that described Shinella zoogloeoides BC026CGMCC № .2224 cultivates in sewage is 30 ℃, and pH value condition is pH8.
7, be the bacteria agent of activeconstituents with Shinella zoogloeoides BC026 CGMCC № .2224.
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