CN106119151A - Bacterial strain and method for screening and separating thereof for degraded 2,4,6 trichlorophenol, 2,4,6,-Ts - Google Patents
Bacterial strain and method for screening and separating thereof for degraded 2,4,6 trichlorophenol, 2,4,6,-Ts Download PDFInfo
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Abstract
For degraded 2, the method for screening and separating of the bacterial strain of 4,6 trichlorophenol, 2,4,6,-Ts, includes the processing technology field of organic pollutants, this this method is by gathering the activated sludge in paper waste internal-circulation anaerobic reactor, in strictly anaerobic environment, carry out acclimating, then acclimation sludge is inoculated in respectively by dilution gradient and is added with 2,4, in the solid screening culture medium of 6 trichlorophenol, 2,4,6,-Ts, isolated is multiple to 2, and 4,6 trichlorophenols have the purification bacterial strain of degradation capability；Finally being inoculated in respectively by multiple purification bacterial strain and be added with 2, in the degraded culture medium of 4,6 trichlorophenol, 2,4,6,-Ts, screening obtains 2, the bacterial strain that the degradation efficiency of 4,6 trichlorophenol, 2,4,6,-Ts is the highest.The method of the present invention is simple, and the purity of the spindle lysine bacillus cereus obtained is higher, and yield is the highest.The present invention also provides for a kind of bacterial strain for 2,4,6 trichlorophenol, 2,4,6,-Ts of degrading, and this bacterial strain can under anaerobic pass through reduction dechlorination degrading chlorophenol type organic, good degrading effect.
Description
Technical field
The present invention relates to the processing technology field of organic pollution, and particularly to one for degraded 2,4,6-trichloro-benzenes
The bacterial strain of phenol and method for screening and separating thereof.
Background technology
Chlorophenol is the chlorinated hydrocarbon for non-agricultural parasite killing, insecticide or antimicrobial class reagent, chlorine
The concentration threshold that phenol produces toxic action to human body is 40ppm, and the threshold value that Fish produce toxic action is only 1ppm, therefore,
Chlorophenol easily causes the pollution of water body, is also classified as priority pollutant and China's key pollutants by U.S. EPA.
At present the processing method of parachlorophenol class material mainly has ultrafiltration, chemical precipitation method, a bioanalysis, wherein ultrafiltration and
Chemical precipitation method exists that energy consumption is big, cost is high, adding of agent easily causes the shortcoming of secondary pollution, and biologic treating technique becomes degraded
The preferable selection of this hardly degraded organic substance of chlorophenol, is primarily present the biology of following three kinds of degradation treatment chlorophenols:
People have utilized bioreactor domestication and have turned out the anaerobic sludge with degrading chlorophenol function, but the party
Method acclimation period is longer, and the anaerobic sludge obtained unblooded chlorophenol degradation bacterium, the most not through primary dcreening operation and multiple sieve, purity
Relatively low, the degradation treatment effect of the most this anaerobic sludge parachlorophenol is unstable.
Meanwhile, more about the biodegradable report of chlorophenol both at home and abroad, isolated is multiple has chlorophenol degradation efficiency
Antibacterial, mainly has pseudomonas (Psendomonas sp.), bacillus cereus (Bacillus sp), dehalogenation desulfiting bacterium
(Desulfitobacterium sp.), greedy copper bacterium (Cupriavidus sp.), actinomycetes (Actinobacteria sp.) etc.
Microbial bacteria, the especially research report of pseudomonas degrading chlorophenol type organic are more.But these microbial bacteria parachlorophenol
Tolerable concentration is the most relatively low, and mostly needs chlorophenols of degrading under aerobic condition, and the degradation treatment effect of Pyrogentisinic Acid depends on
The most poor.
The Chinese invention patent of Application No. 201410853541.9 discloses a kind of paper waste dirt and fills reed Tanaka typical case
Screening of antibacterial spindle lysine bacillus cereus and application thereof, this antibacterial can survive also in dirt fills reed field paper waste
There are good oxygen demand CODcr and the degradation efficiency of absorbable halogenide (Absorbable Organic Halogen, AOX),
The facultative aerobic alkaline-resisting antibacterial of reed field paper waste can be filled as advantage degraded dirt.This bacterial strain through physio-biochemical characteristics and
16SrDNA Analysis and Identification is the bacterial strain that Lysinibacillus belongs to, named LysinibacillusWTXJ1-4；This bacterium carries
Hand over to China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, preservation address: Beijing is exposed to the sun
North Star West Road, district 1 institute 3, preserving number is CGMCCNo.10053.
In sum, spindle lysine bacillus cereus is under anaerobic organic by reduction dechlorination degrading chlorophenol class
Thing, good degrading effect, but the screening technique at present about this bacterial strain screens the spindle lysine bacillus cereus purity obtained
Relatively low, yield is relatively low, it is impossible to meet the extensive process demand of parachlorophenol class material.
Summary of the invention
It is an object of the invention to provide the method for screening and separating of a kind of bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts, side
Method is simple, and the purity of the spindle lysine bacillus cereus obtained is higher, and yield is the highest.
Another object of the present invention is to provide a kind of bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts and, this bacterial strain can be
By reduction dechlorination degrading chlorophenol type organic under anaerobic condition, good degrading effect.
The present invention solves it and technical problem is that and realize by the following technical solutions.
The present invention proposes the method for screening and separating of a kind of bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-T, and it includes following step
Rapid:
Domestication: gather the activated sludge in the internal-circulation anaerobic reactor for processing paper waste, take same volume
Activated sludge and domestication culture medium composition domestication system, the pH of regulation domestication system is 7.0-7.2, by the domestication body after regulation pH
Tie up in anaerobic environment, in 30-40 DEG C, be added with 2, the solid medium of 4,6-trichlorophenol, 2,4,6,-Ts carries out acclimating, directly
In solid medium to domestication, the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts reaches more than 800mg/L, obtains acclimation sludge；
Purification: acclimation sludge is diluted to multiple diluents of variable concentrations, multiple diluents are inoculated in interpolation respectively
Having the 2 of 8-12mg/L, in the solid screening culture medium of 4,6-trichlorophenol, 2,4,6,-Ts, isolated is multiple to 2, and 4,6-trichlorophenols have fall
The purification bacterial strain of solution ability；
Screening: multiple purification bacterial strain is inoculated in respectively the degraded training of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 90-110mg/L
Supporting in base, screening obtains 2, the purification bacterial strain that the degradation efficiency of 4,6-trichlorophenol, 2,4,6,-Ts is the highest, is for degraded 2,4,6-tri-
The bacterial strain of chlorophenol.
Further, in present pre-ferred embodiments, the component of domestication culture medium includes: the K of 1.73g/L2HPO4,
The NH of 1.0g/L4The MgSO of Cl, 0.1g/L4·7H2The CaCl of O, 0.02g/L2, the MnCl of 0.04g/L2·4H2O, 0.05g/L's
FeCl3·6H2The trace element solution of O, 5ml, surplus is sterile distilled water, and regulation pH is 7.0-7.2；Solid screening culture medium
It is the agar being added with 15-20g/L on the basis of domestication culture medium；The component of degraded culture medium and the component of domestication culture medium
Identical.
Further, in present pre-ferred embodiments, it is achieved the method for anaerobic environment is: by the domestication body after regulation pH
System adds extremely tames in culture bottle, is full of nitrogen, then tames cultivation by the rubber stopper seal of band airway in domestication culture bottle
The bottleneck of bottle, makes airway be positioned at one end insertion domestication system of bottle, and hydroxide is inserted in one end that airway is positioned at outside bottle
Sodium solution carries out fluid-tight.
Further, in present pre-ferred embodiments, the method carrying out acclimating is:
Domestication system is placed in anaerobic biological incubator and cultivates, be transferred to add by the inoculum concentration of 10% by culture
In solid medium added with the 2 of 50mg/L, 4,6-trichlorophenol, 2,4,6,-Ts, and put back to continuation cultivation in anaerobic biological incubator, be
One cycle；
The culture continuing to cultivate is cultivated multiple cycle the most continuously, is stepped up in the solid medium in the corresponding cycle
The concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts；
Timing sampling in above-mentioned incubation, the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts in the sample that mensuration obtains, until sample
In product, the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts reaches more than 800mg/L, and the culture that sample is corresponding is acclimation sludge.
Further, in present pre-ferred embodiments, the component of solid medium includes: the Carnis Bovis seu Bubali cream of 5g/L, 10g/L
Peptone, the agar of the NaCl of 5g/L, 20g/L, surplus is water, regulation pH be 7.0, in 121 DEG C of high pressure moist heat sterilizations
20min。
Further, in present pre-ferred embodiments, purification method particularly includes:
By acclimation sludge shaken well, take the acclimation sludge after 1mL vibration successively, become concentration to be 10 by gradient dilution-5's
Diluent, concentration are 10-6Diluent, concentration be 10-7Diluent, taking 0.1mL concentration respectively is 10-5Diluent, concentration
It is 10-6Diluent and concentration be 10-7Diluent, be inoculated on the flat board being loaded with solid screening culture medium, with spreading rod will
Diluent coating is uniformly；
Flat board is placed in anaerobic biological incubator, constant temperature culture at 35 DEG C, after growing bacterium colony on flat board, selects life
Long good bacterium colony, with the inoculation line on another clean flat board of plate streaking partition method, is placed in anaerobic biological by cleaning flat board
Incubator is cultivated, i.e. completes a plate streaking and separate；
Repeating repeatedly plate streaking to separate, until separating, acquisition is multiple has 2,4,6-trichlorophenol, 2,4,6,-T degradation capabilities
Cultivate bacterial strain, multiple cultivation bacterial strain is put in the ultra cold storage freezer of-70 DEG C and preserve.
Further, in present pre-ferred embodiments, screening method particularly includes: respectively multiple cultivation bacterial strain is prepared
Become bacteria suspension, be seeded to bacteria suspension respectively degrade in culture medium, detect in postvaccinal degraded culture medium 2,4,6-tri-respectively
The concentration of chlorophenol, to 2, the cultivation bacterial strain that the degradation efficiency of 4,6-trichlorophenol, 2,4,6,-Ts is the highest, it is for degraded 2,4,6-trichlorines
The bacterial strain of phenol.
Further, in present pre-ferred embodiments, by the method for bacterial suspension inoculation to degraded culture fluid it is: inhale respectively
The bacteria suspension taking 10mL is added with in the degraded culture medium of 2,4,6-trichlorophenol, 2,4,6,-Ts being added with 90-110mg/L of 190mL,
In 35 DEG C of constant temperature culture 5 days.
Further, in present pre-ferred embodiments, the compound method of bacteria suspension is: choose from solid screening culture medium
Take purification strain be inoculated into activation enrichment medium in, constant temperature culture 24h at 35 DEG C, by medium centrifugal 10min, pour out from
Upper liquid after the heart, collects thalline therein, after sterilized water cyclic washing 3 times, is configured to bacteria suspension with sterilized water, wherein,
The component of activation enrichment medium includes: the Carnis Bovis seu Bubali cream of 5g/L, the peptone of 10g/L, the NaCl of 5g/L, and surplus is water, regulation
PH is 7.0, in 121 DEG C of high pressure moist heat sterilization 20min.
A kind of bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-T, uses above-mentioned method for screening and separating to obtain.
The bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-T of the embodiment of the present invention and the beneficial effect of method for screening and separating thereof
It is: this method is by gathering the activated sludge in the internal-circulation anaerobic reactor processing paper waste, at strictly anaerobic
Environment carries out acclimating, then acclimation sludge is inoculated in respectively by dilution gradient and is added with 2, the 4,6-tri-of 8-12mg/L
In the solid screening culture medium of chlorophenol, isolated is multiple to 2, and 4,6-trichlorophenols have the purification bacterial strain of degradation capability；?
After multiple described purification bacterial strain is inoculated in the degraded culture medium of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 90-110mg/L respectively,
Screening obtains 2, the bacterial strain that the degradation efficiency of 4,6-trichlorophenol, 2,4,6,-Ts is the highest.The method is simple, the spindle lysine bud obtained
The purity of spore bacillus is higher, and yield is the highest, and this bacterial strain can under anaerobic pass through reduction dechlorination degrading chlorophenol type organic, fall
Solve effective.
Accompanying drawing explanation
In order to be illustrated more clearly that the technical scheme of the embodiment of the present invention, below by embodiment required use attached
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, and it is right to be therefore not construed as
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to according to this
A little accompanying drawings obtain other relevant accompanying drawings.
Fig. 1 is the strain of variable concentrations in the embodiment of the present invention and to 2, the linear pass of the degradation rate of 4,6-trichlorophenol, 2,4,6,-Ts
System's figure；
Fig. 2 is the strain of embodiment of the present invention different vaccination amount and in soil 2, the degradation rate of 4,6-trichlorophenol, 2,4,6,-Ts
Linear relationship chart.
Detailed description of the invention
For making the purpose of the embodiment of the present invention, technical scheme and advantage clearer, below will be in the embodiment of the present invention
Technical scheme be clearly and completely described.In embodiment, unreceipted actual conditions person, builds according to normal condition or manufacturer
The condition of view is carried out.Agents useful for same or instrument unreceipted production firm person, being can be by the commercially available conventional product bought and obtain
Product.
The bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-T and method for screening and separating thereof to the embodiment of the present invention are carried out below
Illustrate.
The embodiment of the present invention provides one for the method for screening and separating of the bacterial strain of degraded 2,4,6-trichlorophenol, 2,4,6,-T, and it includes
Following steps:
S1 tames: gathers the activated sludge in the internal-circulation anaerobic reactor for processing paper waste, takes same volume
Activated sludge and domestication culture medium composition domestication system, regulation domestication system pH be 7.0-7.2, by regulation pH after domestication
System is in strictly anaerobic environment, in 30-40 DEG C, is being added with 2, carries out enrichment and tame and docile in the solid medium of 4,6-trichlorophenol, 2,4,6,-Ts
Change, until the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts reaches more than 800mg/L in the solid medium after Xun Hua, obtain acclimation sludge,
Enrichment is to collect the trace element to be measured from a large amount of parent materials to a smaller size smaller, thus improves its content under measuring
This operating procedure that limit is above.Domestication is the material adding targeting environment incremental in bacteria culture media or substrate,
Allow antibacterial gradually adapt to and rely on material or the substrate of targeting environment, thus reaching the effective ingredient improving or changing in environment.
Wherein, the component of domestication culture medium includes: the K of 1.73g/L2HPO4, the NH4Cl of 1.0g/L, 0.1g/L's
MgSO4·7H2The CaCl of O, 0.02g/L2, the MnCl of 0.04g/L2·4H2The FeCl of O, 0.05g/L3·6H2The trace of O, 5ml
Element Solution, surplus is sterile distilled water, and regulation pH is 7.0-7.2.
The component of solid medium includes: the Carnis Bovis seu Bubali cream of 5g/L, the peptone of 10g/L, the fine jade of the NaCl of 5g/L, 20g/L
Fat, surplus is water, and regulation pH is 7.0, in 121 DEG C of high pressure moist heat sterilization 20min.
In above-mentioned steps, internal-circulation anaerobic reactor includes the mixed zone from bottom to top arranged, the first anaerobic zone, second detests
Oxygen district, settling zone and gas-liquid separation zone, activated sludge is taken from the first anaerobic zone.
The concrete grammar realizing strictly anaerobic environment is: be added with the domestication system after regulation pH to domestication culture bottle
In, in domestication culture bottle, it is full of nitrogen, then the bottleneck with the rubber stopper seal domestication culture bottle of band airway, makes airway position
One end in bottle is inserted in domestication system, and one end that airway is positioned at outside bottle is inserted in sodium hydroxide solution and carried out fluid-tight.
The concrete grammar carrying out acclimating in above-mentioned steps is:
Domestication system is placed in anaerobic biological incubator and cultivates, be transferred to add by the inoculum concentration of 10% by culture
In solid medium added with the 2 of 50mg/L, 4,6-trichlorophenol, 2,4,6,-Ts, and put back to continuation cultivation in anaerobic biological incubator, above-mentioned
Process is a cycle.
The culture continuing to cultivate is cultivated multiple cycle the most continuously, is stepped up in the solid medium in the corresponding cycle
The concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts.
Timing sampling in above-mentioned incubation, the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts in the sample that mensuration obtains, until sample
In product, the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts reaches more than 800mg/L, and the culture that sample is corresponding is acclimation sludge.
S2 purification: acclimation sludge is inoculated in respectively by dilution gradient 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 8-12mg/L
In solid screening culture medium, isolated is multiple to 2, and 4,6-trichlorophenols have the purification bacterial strain of degradation capability.Wherein, solid sieve
Selecting culture medium is to be added with the agar of 15-20g/L on the basis of domestication culture medium, and the component of solid screening culture medium includes:
The agar of 15-20g/L, the K of 1.73g/L2HPO4, the MgSO of the NH4Cl of 1.0g/L, 0.1g/L4·7H2O, 0.02g/L's
CaCl2, the MnCl of 0.04g/L2·4H2The FeCl of O, 0.05g/L3·6H2The trace element solution of O, 5ml, surplus is aseptic steaming
Distilled water, regulation pH is 7.0-7.2.
Above-mentioned purification method particularly includes:
By acclimation sludge shaken well, take the acclimation sludge after 1mL vibration successively, become concentration to be 10 by gradient dilution-3's
Diluent, concentration are 10-4Diluent, concentration be 10-5Diluent, concentration be 10-6Diluent, concentration be 10-7Dilution
Liquid, taking 0.1mL concentration respectively is 10-5Diluent, concentration be 10-6Diluent and concentration be 10-7Diluent, be inoculated into
It is loaded with on the flat board of solid screening culture medium, with spreading rod by diluent coating uniformly.
Flat board is placed in anaerobic biological incubator, constant temperature culture at 35 DEG C, after growing bacterium colony on flat board, selects life
Long good bacterium colony, with the inoculation line on another clean flat board of plate streaking partition method, is placed in anaerobic biological by cleaning flat board
Incubator is cultivated, i.e. completes a plate streaking and separate.
Repeating repeatedly plate streaking to separate, until separating, acquisition is multiple has 2,4,6-trichlorophenol, 2,4,6,-T degradation capabilities
Cultivate bacterial strain, multiple cultivation bacterial strain is put in the ultra cold storage freezer of-70 DEG C and preserve.
S3 screens: multiple purification bacterial strain is inoculated in the degraded of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 90-110mg/L respectively
In culture medium, screening obtains 2, the described purification bacterial strain that the degradation efficiency of 4,6-trichlorophenol, 2,4,6,-Ts is the highest, is for degraded 2,
The bacterial strain of 4,6-trichlorophenol, 2,4,6,-Ts.Wherein, the component of degraded culture medium is identical with the component of domestication culture medium.The group of degraded culture medium
Divide and include: the K of 1.73g/L2HPO4, the MgSO of the NH4Cl of 1.0g/L, 0.1g/L4·7H2The CaCl of O, 0.02g/L2, 0.04g/L
MnCl2·4H2The FeCl of O, 0.05g/L3·6H2The trace element solution of O, 5ml, surplus is sterile distilled water, and regulation pH is
7.0-7.2。
Above-mentioned screening method particularly includes: respectively multiple cultivation bacterial strain is configured to bacteria suspension, bacteria suspension is inoculated respectively
To degraded culture medium, detect the dense of 2,4,6-trichlorophenol, 2,4,6,-Ts in postvaccinal degraded culture medium by high performance liquid chromatography respectively
Degree, the cultivation bacterial strain in the degraded culture medium that the degradation efficiency of 2,4,6-trichlorophenol, 2,4,6,-Ts is the highest is for 2,4,6-trichlorines of degrading
The bacterial strain of phenol.
The compound method of bacteria suspension is: from solid screening culture medium, picking purification strain is inoculated into activation enrichment medium
In, constant temperature culture 24h at 35 DEG C, by medium centrifugal 10min, pour out the upper liquid after being centrifuged, collect thalline therein, use
After sterilized water cyclic washing 3 times, being configured to bacteria suspension with sterilized water, wherein, the component of activation enrichment medium includes: 5g/L's
Carnis Bovis seu Bubali cream, the peptone of 10g/L, the NaCl of 5g/L, surplus is water, and regulation pH is 7.0, in 121 DEG C of high pressure moist heat sterilizations
20min。
By the method for bacterial suspension inoculation to degraded culture fluid it is: the bacteria suspension drawing 10mL respectively is added with 190mL's
It is added with the 2 of 90-110mg/L, in the degraded culture medium of 4,6-trichlorophenol, 2,4,6,-Ts, in 35 DEG C of constant temperature culture 5 days.
The embodiment of the present invention also provides for a kind of bacterial strain for 2,4,6-trichlorophenol, 2,4,6,-Ts of degrading, and uses above-mentioned screening and separating
Method obtains.
Below in conjunction with embodiment, inventive feature and performance are described in further detail.
Embodiment 1
Gather certain paper mill activated sludge in the internal-circulation anaerobic reactor processing paper waste, measure 1L semi-fluid
The activated sludge of body shape adds to domestication culture bottle, adds 1L and tames culture medium, regulates pH to 7.0, after regulation pH
Domestication system, in strictly anaerobic environment, carries out acclimating at 30 DEG C, until 2,4,6-in the domestication system after Xun Hua
The concentration of trichlorophenol, 2,4,6,-T reaches 800mg/L, obtains acclimation sludge.
Acclimation sludge is inoculated in respectively by dilution gradient the solid screening training of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 8mg/L
Supporting in base, isolated 3 kinds is to 2, and 4,6-trichlorophenols have a purification bacterial strain of degradation capability: A1, A2, A3.
Purification strains A 1, A2, A3 are inoculated in respectively the degraded culture medium of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 90mg/L
In, after testing, purification strains A 1 is to 2, and the degradation efficiency of 4,6-trichlorophenol, 2,4,6,-Ts is the highest, and purification strains A 1 is for degraded 2,4,
The bacterial strain of 6-trichlorophenol, 2,4,6,-T.
Embodiment 2
Gather certain paper mill activated sludge in the internal-circulation anaerobic reactor processing paper waste, measure 1L semi-fluid
The activated sludge of body shape adds to domestication culture bottle, adds 1L and tames culture medium, regulates pH to 7.2, after regulation pH
Domestication system, in strictly anaerobic environment, carries out acclimating at 40 DEG C, until 2,4,6-in the domestication system after Xun Hua
The concentration of trichlorophenol, 2,4,6,-T reaches 800mg/L, obtains acclimation sludge.
Acclimation sludge is inoculated in respectively by dilution gradient the solid screening of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 12mg/L
In culture medium, isolated 5 kinds is to 2, and 4,6-trichlorophenols have a purification bacterial strain of degradation capability: C1, C2, C3, C4, C5.
Purification bacterial strain C1, C2, C3, C4, C5 are inoculated in respectively the degraded of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 110mg/L
In culture medium, after testing, purification bacterial strain C2 is to 2, and the degradation efficiency of 4,6-trichlorophenol, 2,4,6,-Ts is the highest, and purification bacterial strain C2 is for dropping
Solve 2,4, the bacterial strain of 6-trichlorophenol, 2,4,6,-T.
Embodiment 3
Gathering activated sludge in certain waste water of paper mill internal circulating anaerobic treatment reactor, the activity measuring 1L semifluid shape is dirty
Mud adds to domestication culture bottle, adds 1L and tames culture medium, regulates pH to 7.0.Nitrogen it is full of in domestication culture bottle, then
Tame the bottleneck of culture bottle by the rubber stopper seal of band airway, domestication system is inserted in the one end making airway be positioned at bottle,
One end that airway is positioned at outside bottle is inserted in sodium hydroxide solution and is carried out fluid-tight.The whole series domesticating device is placed in anaerobic biological cultivate
Case is cultivated at 30-40 DEG C.Culture is transferred to be added with the 2 of 50mg/L, 4,6-trichlorines by the inoculum concentration of 10%
In the solid medium of phenol, and put back to continuation cultivation in anaerobic biological incubator, be stepped up solid culture by identical way
The concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts in base, cultivates several cycles continuously, and in domestication incubation, timing sampling measures domestication sample
In the residual concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts, until 2,4,6-trichlorophenol, 2,4,6,-Ts reach 800mg/L, the culture that this sample is corresponding is
Acclimation sludge.
By acclimation sludge shaken well, take the acclimation sludge after 1mL vibration successively, become concentration to be 10 by gradient dilution-3's
Diluent, concentration are 10-4Diluent, concentration be 10-5Diluent, concentration be 10-6Diluent, concentration be 10-7Dilution
Liquid, taking 0.1mL concentration respectively is 10-5Diluent, concentration be 10-6Diluent and concentration be 10-7Diluent, be inoculated into
It is loaded with on the flat board of solid screening culture medium, with spreading rod by diluent coating uniformly.Flat board is placed in anaerobic biological incubator
In, constant temperature culture at 35 DEG C, after growing bacterium colony on flat board, select well-grown bacterium colony, exist by plate streaking partition method
Inoculation line on another clean flat board, repeatedly plate streaking separates, until separate obtain 4 kinds of purification bacterial strains be respectively B1,
These 4 kinds of purification bacterial strains are put in the ultra cold storage freezer of-70 DEG C by H1, DH-1 and 1-4.
In from solid medium, picking purification strain is inoculated into activation enrichment medium, constant temperature culture 24h at 35 DEG C,
By medium centrifugal 10min, pour out the upper liquid after being centrifuged, collect thalline therein, after sterilized water cyclic washing 3 times, use
Sterilized water is configured to bacteria suspension, and absorption 10mL bacteria suspension joins 190mL and is added with the 2 of 100mg/L the most respectively, 4,6-trichlorines
In the degraded culture medium of phenol, in 35 DEG C of constant temperature culture 5 days, with 2,4,6-trichlorophenol, 2,4,6,-Ts in high-performance liquid chromatogram determination solution
Concentration, 4 kinds of strains are as shown in table 1 to the degradation rate of 2,4,6-trichlorophenol, 2,4,6,-Ts in degraded culture medium,
As can be seen from the above table, purification bacterial strain 1-4 is to 2, and it is the highest that 4,6-trichlorophenol, 2,4,6,-Ts connect degradation efficiency, therefore, and purification bacterium
Strain 1-4 is the bacterial strain for 2,4,6-trichlorophenol, 2,4,6,-Ts of degrading.
The physiological feature for the bacterial strain of degraded 2,4,6-trichlorophenol, 2,4,6,-Ts obtained embodiment 2 below detects.
1, morphological characteristic
(1) colony morphology characteristic: white, opaque, neat in edge, circular, smooth surface and slightly swelling.
(2) somatic cells morphological characteristic: this bacterium is gram positive bacteria.
2, the 16S rRNA sequence analysis of bacterial strain:
The PCR amplification of test strain 16S rRNA and sequencing: using the DNA extracted as expanding template.16SrRNA expands
The forward primer Pf:5 '-AGAGTTTGATCCTGGCTCAG-3 ' used by PCR reaction increased；Reverse primer Pr:5 '-
GGTTACCTTGTTACGA CTT-3 ', corresponds respectively to 8-27 and the 1492-1510 base of the 16S rRNA gene of bacterium.
PCR reaction system (50 μ L) is: template 2 μ L, dNTP (25mmol/L) 5 μ L, each 1 μ L of primer (1mmol/L), 10 ×
PCR buffer 5 μ L, Mg2+ (25mmol/L) 4 μ L, Taq enzyme (5U/ μ L) 0.4 μ L, ultra-pure water 31.6 μ L.
PCR reaction condition: 94 DEG C, 4min；94 DEG C, 1min；54 DEG C, 1min；72 DEG C of 1.5min, circulate 30 times；72 DEG C are prolonged
Stretch 7min.16S rRNA amplification and sequencing are completed by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, the survey obtained
Sequence result has been uploaded to Genbank (accession number is KP150574).From GeneBank, similarity is recalled with Blast search utility
The 16S rRNA gene order of higher related strain, carries out Multiple Sequence Alignment with CLUSTAL1.8, according to Kimura 2-
Parameter model assessment phyletic evolution distance matrix, uses adjacent method (Neighbor-Joining) with MEGA3.0 software kit
Carry out cluster analysis, finally determine that this bacterial strain (purification strain 1-4) is capitate lysine bacillus cereus Lysinibacillus
Fusiformis WTXJ1-4 (CGMCC preserving number: No.10053).
3, bacterial strain is to 2, the degradation effect of 4,6-trichlorophenol, 2,4,6,-Ts
Preparing a series of containing variable concentrations 2, the minimal medium of 4,6-trichlorophenol, 2,4,6,-Ts, regulation pH is 7.0；Take 10mL to join
The bacteria suspension made joins in 190mL inorganic salt, it is respectively 50 containing concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts, 100,200,400,
600mg/L；Then by the inorganic salt added with bacteria suspension at 35 DEG C, cultivate under anaerobic condition, periodically sample detection, each sample
If 2 repetitions, and make blank to be not added with the inorganic salt of strain, the strain of variable concentrations to 2, the degraded of 4,6-trichlorophenol, 2,4,6,-Ts
The result of rate such as Fig. 1.
As shown in Figure 1, the initial concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts has extreme influence to the degradation property of bacterial strain: 2,4,6-tri-
When chlorophenol is between 50-600mg/L, its degradation rate is all less than 60%, especially when high concentration, such as 600mg/L, and 2,4,6-tri-
The degradation rate of chlorophenol is less than 5%.2,4,6-trichlorophenol, 2,4,6,-T concentration are 50mg/L, are more beneficial for 2,4,6-trichlorophenol, 2,4,6,-Ts
Degraded, degradation rate reaches 60%.
3, bacterial strain is in soil 2, the degradation effect of 4,6-trichlorophenol, 2,4,6,-Ts
Weigh a series of 20g soil in 250mL conical flask, add 0,2,1% (every 100mL water bacterial strain Han 1g) of 4mL
Bacteria suspension, bottleneck tampon stoppers, Anaerobic culturel in 35 DEG C of calorstats, the pollution concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts in soil
For 50mg/kg, holding water content is saturation moisture content the 60% of soil, and the maintenance constant weight that adds water every day all the time, the 0th, 5,
10,20,30,40 days sample analysis, the strain of different vaccination amount in soil 2, the degradation rate of 4,6-trichlorophenol, 2,4,6,-Ts such as Fig. 2 institute
Show.
As shown in Figure 2, the bacteria suspension of addition 4mL is in soil 2, and the degradation rate of 4,6-trichlorophenol, 2,4,6,-Ts is maximum, can reach
60%.
In sum, the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-T of the embodiment of the present invention and method for screening and separating thereof,
The method is simple, and the purity of the spindle lysine bacillus cereus obtained is higher, and yield is the highest, and this bacterial strain can be under anaerobic
By reduction dechlorination degrading chlorophenol type organic, good degrading effect.
Embodiments described above is a part of embodiment of the present invention rather than whole embodiments.The reality of the present invention
The detailed description executing example is not intended to limit the scope of claimed invention, but is merely representative of the selected enforcement of the present invention
Example.Based on the embodiment in the present invention, those of ordinary skill in the art are obtained under not making creative work premise
Every other embodiment, broadly falls into the scope of protection of the invention.
Claims (10)
1. the method for screening and separating for the bacterial strain of degraded 2,4,6-trichlorophenol, 2,4,6,-T, it is characterised in that it includes following step
Rapid:
Domestication: gather the activated sludge in the internal-circulation anaerobic reactor for processing paper waste, take the described of same volume
Activated sludge and domestication culture medium composition domestication system, regulation domestication system pH be 7.0-7.2, will regulation pH after described tame and docile
Change system is in anaerobic environment, in 30-40 DEG C, is being added with 2, carries out enrichment and tame and docile in the solid medium of 4,6-trichlorophenol, 2,4,6,-Ts
Change, until the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts reaches more than 800mg/L in the described solid medium after Xun Hua, obtain domestication dirt
Mud；
Purification: described acclimation sludge is diluted to multiple diluents of variable concentrations, the plurality of diluent is inoculated in respectively
Being added with the 2 of 8-12mg/L, in the solid screening culture medium of 4,6-trichlorophenol, 2,4,6,-Ts, isolated is multiple to 2,4,6-trichlorophenol tools
There is the purification bacterial strain of degradation capability；
Screening: multiple described purification bacterial strain is inoculated in respectively the degraded training of 2,4, the 6-trichlorophenol, 2,4,6,-Ts being added with 90-110mg/L
Supporting in base, screening obtains 2, the described purification bacterial strain that the degradation efficiency of 4,6-trichlorophenol, 2,4,6,-Ts is the highest, is for degraded 2,4,
The bacterial strain of 6-trichlorophenol, 2,4,6,-T.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 1, its feature exists
In, the component of described domestication culture medium includes: the K of 1.73g/L2HPO4, the NH of 1.0g/L4The MgSO of Cl, 0.1g/L4·7H2O,
The CaCl of 0.02g/L2, the MnCl of 0.04g/L2·4H2The FeCl of O, 0.05g/L3·6H2The trace element solution of O, 5ml, surplus
For sterile distilled water, regulation pH is 7.0-7.2；Described solid screening culture medium is to add on the basis of described domestication culture medium
There is the agar of 15-20g/L；The component of described degraded culture medium is identical with the component of described domestication culture medium.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 1, its feature exists
In, it is achieved the method for described anaerobic environment is: the described domestication system after regulation pH added to domestication culture bottle, to described
Domestication culture bottle is full of nitrogen, then with taming the bottleneck of culture bottle described in the rubber stopper seal of band airway, makes described inducing QI
Pipe is positioned at one end of bottle and inserts described domestication system, and one end that described airway is positioned at outside bottle is inserted in sodium hydroxide solution
Carry out fluid-tight.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 1, its feature exists
In, the method carrying out acclimating is:
Described domestication system is placed in anaerobic biological incubator and cultivates, be transferred to add by the inoculum concentration of 10% by culture
In described solid medium added with the 2 of 50mg/L, 4,6-trichlorophenol, 2,4,6,-Ts, and put back to continuation training in described anaerobic biological incubator
Support, be a cycle；
The described culture continuing to cultivate is cultivated multiple described cycle the most continuously, is stepped up the described solid in the corresponding cycle
The concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts in culture medium；
Timing sampling in above-mentioned incubation, the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts in the sample that mensuration obtains, until described sample
In product, the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts reaches more than 800mg/L, and the described culture that described sample is corresponding is described domestication dirt
Mud.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 1, its feature exists
In, the component of described solid medium includes: the Carnis Bovis seu Bubali cream of 5g/L, the peptone of 10g/L, the fine jade of the NaCl of 5g/L, 20g/L
Fat, surplus is water, and regulation pH is 7.0, in 121 DEG C of high pressure moist heat sterilization 20min.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 1, its feature exists
In, described purification method particularly includes:
By described acclimation sludge shaken well, take the described acclimation sludge after 1mL vibration successively, become concentration to be 10 by gradient dilution-5Diluent, concentration be 10-6Diluent, concentration be 10-7Diluent, taking concentration described in 0.1mL respectively is 10-5Dilution
Liquid, described concentration are 10-6Diluent and described concentration be 10-7Diluent, be inoculated into and be loaded with described solid screening culture medium
Flat board on, with spreading rod by the coating of described diluent uniformly；
Described flat board is placed in anaerobic biological incubator, constant temperature culture at 35 DEG C, after growing bacterium colony on described flat board, chooses
Select well-grown described bacterium colony, with the inoculation line on another clean flat board of plate streaking partition method, by described clean flat board
It is placed in described anaerobic biological incubator cultivation, i.e. completes a plate streaking and separate；
Repeating the most described plate streaking to separate, until separating, acquisition is multiple has 2,4,6-trichlorophenol, 2,4,6,-T degradation capabilities
Cultivate bacterial strain, multiple described cultivation bacterial strain is put in the ultra cold storage freezer of-70 DEG C and preserve.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 6, its feature exists
In, described screening method particularly includes: respectively multiple described cultivation bacterial strain is configured to bacteria suspension, described bacteria suspension is connect respectively
Plant to described degraded culture medium, detect the concentration of 2,4,6-trichlorophenol, 2,4,6,-Ts in postvaccinal described degraded culture medium respectively, right
The described cultivation bacterial strain that the degradation efficiency of 2,4,6-trichlorophenol, 2,4,6,-Ts is the highest, is the described bacterium for 2,4,6-trichlorophenol, 2,4,6,-Ts of degrading
Strain.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 7, its feature exists
In, by the method for bacterial suspension inoculation to degraded culture fluid it is: the described bacteria suspension drawing 10mL respectively is added with adding of 190mL
In described degraded culture medium added with the 2 of 90-110mg/L, 4,6-trichlorophenol, 2,4,6,-Ts, in 35 DEG C of constant temperature culture 5 days.
The method for screening and separating of the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-Ts the most according to claim 7, its feature exists
In, the compound method of described bacteria suspension is: from described solid screening culture medium, purification strain described in picking is inoculated into activation richness
In collection culture medium, constant temperature culture 24h at 35 DEG C, by medium centrifugal 10min, pour out the upper liquid after being centrifuged, collect wherein
Thalline, after sterilized water cyclic washing 3 times, be configured to described bacteria suspension, wherein, described activation enrichment medium with sterilized water
Component include: the Carnis Bovis seu Bubali cream of 5g/L, the peptone of 10g/L, the NaCl of 5g/L, surplus is water, regulation pH be 7.0, in 121 DEG C
High pressure moist heat sterilization 20min.
10. the bacterial strain for degraded 2,4,6-trichlorophenol, 2,4,6,-T, it is characterised in that use institute any one of claim 1 to 9
The method for screening and separating stated obtains.
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| CN114015637A (en) * | 2021-11-29 | 2022-02-08 | 浙江海洋大学 | Screening method and application of benzoyl dimethyl ammonium compound degradation bacterium groups |
| CN116496931A (en) * | 2022-12-30 | 2023-07-28 | 陕西省微生物研究所 | Enterobacter cloacae, preparation method thereof, culture medium and application |
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