CN106244499A - One strain Di Monei Marseille bacterium new strains and application thereof - Google Patents
One strain Di Monei Marseille bacterium new strains and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D3/00—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances
- A62D3/02—Processes for making harmful chemical substances harmless or less harmful, by effecting a chemical change in the substances by biological methods, i.e. processes using enzymes or microorganisms
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- A—HUMAN NECESSITIES
- A62—LIFE-SAVING; FIRE-FIGHTING
- A62D—CHEMICAL MEANS FOR EXTINGUISHING FIRES OR FOR COMBATING OR PROTECTING AGAINST HARMFUL CHEMICAL AGENTS; CHEMICAL MATERIALS FOR USE IN BREATHING APPARATUS
- A62D2101/00—Harmful chemical substances made harmless, or less harmful, by effecting chemical change
- A62D2101/20—Organic substances
Abstract
The invention discloses a strain new strains Di Monei Marseille bacterium J42, and this bacterial strain is as oil degradation bacteria application in degraded oil waste water；This bacterial strain is isolated one strain new strains Di Monei Marseille bacterium J42 from Xiang River dredger district bed mud, and this bacterial strain not only has good biosurfactant production ability, and this bacterial strain has stronger emulsifying activity and reduces surface tension of liquid ability；It addition, J42 fungus beetle benzene degradation experiment shows that this bacterial strain has the ability of extremely strong degraded aromatic hydrocarbon toluene；This bacterial strain also shows stronger COD removal efficiency in degraded oil waste water is tested, and the present invention demonstrates that this bacterial strain has a good application prospect in oil pollution biological restoration field.
Description
Technical field
The present invention relates to a strain Di Monei Marseille bacterium new strains, further relate to this Di Monei Marseille bacterium new strains in oil pollution
Purposes in biological restoration, belongs to biological technical field.
Background technology
Oil belongs to inflammable minerals matter, is the earth different periods of history in formation, by the Organic substance such as plant or animal
Remains generate.Alkane is composition stone main body of oil, and with the increase of relative molecular mass, alkane is respectively with gas, liquid, solid three
State is present in oil.Aromatic hydrocarbon accounts for the 2-4% of oil content, has monocycle class (such as benzene,toluene,xylene), possibly together with dicyclo
(mainly naphthalene), thrcylic aromatic hydrocarbon (such as phenanthrene) and the above polycyclic hydrocarbon of three rings.
Biological toxicity can be divided into two classes, a class to be the acute poisonings that bulk petroleum causes by oil；Another kind of is long-term
The poisonous effect of low concentration oil.The refining oil product toxicity of general light oil is bigger than crude oil, the toxicity of oil and oil product with
The content of the solubility arene derivatives (benzene, naphthalene, phenanthrene etc.) wherein contained is proportional.Oil poisonous effect in water body
Most normal alkane low from the relative molecular mass that water solublity is big and mononuclear aromatics.Wherein aromatic hydrocarbons is maximum to biological toxicity.
At present in the improvement of oil pollution, the technology taked has physics, chemistry and biological method, and wherein physical method is
Petroleum hydrocarbon is diluted, assembles or is moved in other environment, and chemical method is difficult to thoroughly degrade petroleum hydrocarbon, and
Chemical method can cause secondary pollution.By contrast, biological restoration has that safety, economy be strong, applied range, removal efficiency
High and without remarkable advantages such as obvious secondary pollutions.
Many studies have shown that, biosurfactant has good facilitation in oil degradation.Different microorganisms is past
Toward the surfactant of generation different structure, mainly there are glycolipid, lipopeptid, polysaccharide-protein complex, phospholipid, fatty acid and neutrality
Fat etc..On the one hand biosurfactant can increase the surface area of non-water soluble substance, improve the dissolubility of Hydrocarbon
Thus beneficially biological utilisation；On the other hand surfactant can produce substantial amounts of biological emulsifier thus contribute to biodegradation
Process.This can be greatly improved the energy for growth of degradable oil antibacterial and improve the utilization to petroleum hydrocarbon of these antibacterials.
Finding the capillary minimizing of stone oil-water system that bacterial growth metabolism can cause, capillary reduction can increase simultaneously
Oil dissolubility in water and dispersive property, thus add the contact surface area of oil-water and dispersion degree and thin
Bacterium and the contact area of oil, have facilitation to the oil degradation of antibacterial.And temperature, pH are closely related with the degraded of oil,
Because it can directly affect growth and the activity of microorganism.
Therefore this seminar is by Xiang River dredger region bed mud strain screening, is desirably to obtain and has good generation thing
Surfactant abilities, emulsifying activity, reduction surface tension of liquid ability and the higher new strains of petroleum degradation rate, for degraded stone
Oil contaminants repairing environment provides more efficient more bacterium source.
Summary of the invention
It is an object of the invention to provide and a kind of there is good surfactant abilities, emulsifying activity, reduction liquid surface
Tension force ability and there is the new strains J42 of oil degradation ability.
Another object of the present invention is that the new strains proposing described Di Monei Marseille Pseudomonas is in repairing pollution environment
Application.
For realizing the purpose of foregoing invention, the technical scheme that the present invention takes is as follows:
A kind of new bacterium that there is good biosurfactant production ability, emulsifying activity and there is oil degradation ability
Strain, described new strains is entitled: J42；
Described J42 fungus strain separates from Xiang River dredger region bed mud, screening, purification and cultivating, through morphology and point
Sub-Biology identification, determines that this bacterial strain is Di Monei Marseille Pseudomonas, with Massiliatimonae strain UR/MT95 homology
Property 96%.
Described B11 bacterium Main Morphology is characterized as: orange-yellow, circular protrusions, neat in edge, moistening.
Compared with prior art, the beneficial effects are mainly as follows: separate from the bed mud of Xiang River dredger region
Obtaining the Di Monei Marseille bacterium-new strains J42 with oil degradation ability that a strain is rare, this bacterial strain not only has good product
Biosurfactant ability and have good emulsifying activity and reduce surface tension of liquid ability.
Accompanying drawing explanation
The present invention will be further described the most in conjunction with the embodiments.
Fig. 1 is toluene level standard curve；
In Fig. 2 culture fluid, toluene level changes over situation；
Detailed description of the invention
Below by specific embodiment, technical scheme is done the brightest, but the present invention is also
It is not limited to these embodiments.
Embodiment 1
1) collection Xiang River dredger district bed mud is as sample, 4 DEG C of Refrigerator stores.Weigh for examination pedotheque 30g access 100ml without
In bacterium water, add 10g bead.Being positioned in the constant-temperature table of 180rpm, vibrate 30min.
2) take out triangular flask after having vibrated, stand 20min at room temperature, take 1ml supernatant, aseptically carry out
10 times of serial dilutions are to 10-7, take 10 respectively-3、10-5、10-7The dilution bacteria suspension 25 μ L of three concentration be coated in advance
It is coated with the yeast solids minimal medium of toluene, is placed in room temperature condition in vacuum desiccator and cultivates 24h, treat that planar surface is not
It is inverted flat board when having water droplet again.
3) time grown according to bacterium colony in yeast culture medium and the difference of the form of bacterium colony, choose from culture medium flat plate
Take single bacterium colony line to cultivate further to solid inorganic salt culture medium.The most streak culture antibacterial is until single bacterium of growing of flat board
The form that falls is completely the same, and single bacterium colony microorganism individual morphology under the microscope is the most completely the same, can confirm that as pure training
Support.
4) the most purified good antibacterial of picking, is inoculated in the liquid inorganic salt culture medium containing toluene respectively.Cultivate early stage
The culture medium of 50ml system needs add the yeast powder solution that 500 μ L concentration are 2% (w/v).After passing on twice, no longer add ferment
Female powder solution, obtaining can be with toluene as sole carbon source, can the purpose bacterial strain of good growth in liquid medium within.
The preparation of the yeast solids minimal medium containing toluene: draw 25 μ L toluene and be transferred to the training of yeast solids inorganic salt
Support on base, and with aseptic spreading rod by its uniform application in media surface.
The preparation of the solid inorganic salt culture medium containing yeast: i.e. many additions in the solid inorganic salt culture medium of 1L system
2.0g carbamide and 1.0g yeast.
Solid inorganic salt culture medium is prepared: add the agar of 1.5% (W/V) in liquid inorganic salt culture medium.
Wherein, liquid inorganic salt culture medium formula is: carbonate buffer solution 1000mL, MgSO4·7H2O0.1g、NH4Cl
0.5g、Na2HPO40.5g、CaCl2·6H2O 0.01g、FeSO4.7H2O 0.005g, trace element 1mL；Wherein, trace element
Formula be: ZnSO4·7H2O 0.44g、CuSO4·5H2O 0.20g、MnSO4·2H2O 0.17g、Na2MoO4·2H2O
0.06g、H3BO30.10g、CoCl2·6H2O 0.08g, it is settled to 1000mL with distilled water；The formula of carbonate buffer solution is:
Na2CO30.105g、NaHCO30.756g, it is settled to 1000mL, pH8.0 with distilled water.
Embodiment 2
The J42 strain morphology of the present invention and molecular biology research.
Wherein, the LB culture medium prescription of the present embodiment is: yeast extract 5.0g, peptone 10.0g, sodium chloride
10.0g, agar 15.0g, pH 7.2-7.4.
The J42 inoculation present invention obtained, on LB solid medium flat board, is inverted under the conditions of 30 DEG C and is cultivated
24h, observes its colonial morphology.
Described new strains morphological feature is: orange-yellow, circular protrusions, neat in edge, and smooth surface moistens.
Strain identification
Take the aseptic EP pipe of 200 μ L, in pipe, add 50 μ L aseptic double-distilled waters, then take the J42 bacterium that a small amount of present invention obtains
Strain thalline is suspended in EP pipe, finally repeatedly inhales with pipettor and beats EP liquid in pipe and make thalline fully mix to make bacteria suspension.Will
Bacteria suspension 99 DEG C of process 5min in PCR instrument obtain breaking cellular wall bacteria suspension.With bacterial 16 S rRNA universal primer (forward primer 27f,
Downstream primer 1492r) 16SrRNA of amplification purification bacterial strain.
The condition of pcr amplification reaction is: 94 DEG C of denaturations 5min, enters circulation, 94 DEG C of degeneration 30S, 72 DEG C of extensions
1.5min, 72 DEG C extend 4 DEG C of states of preservation after 10min.After reaction terminates, pcr amplification product is carried out 1.0% agarose gel
Electrophoresis detection, result obtains the single band of about 1600bp through amplification.From PCR reactant system, reclaim PCR expand bar
Band, delivers to the rich still biological order-checking in Changsha.By sequencing result comparison in ncbi database.The homologous sequence choosing highest scoring is protected
Deposit bacterial strain name and base sequence, for phylogenetic tree construction.
Through order-checking, 16S rDNA and the Massiliatimonae strain UR/MT95 homology of bacterial strain J42 is up to
96%.
Comprehensive morphological is observed and 16S rDNA sequencing result, it may be determined that this new strains J42 is Di Monei Marseille bacterium.
Embodiment 3
The surfactant of bacterial strain produces characteristic research
Liquid culture culture medium prescription used by the present embodiment is: carbonate buffer solution 1000mL, MgSO4·7H2O
0.1g、NH4Cl 0.5g、Na2HPO40.5g、CaCl2·6H2O 0.01g、FeSO4.7H2O0.005g, toluene 5g, trace element
1mL；Wherein, the formula of trace element is: ZnSO4·7H2O 0.44g、CuSO4·5H2O 0.20g、MnSO4·2H2O0.17g、
Na2MoO4·2H2O 0.06g、H3BO30.10g、CoCl2·6H2O 0.08g, it is settled to 1000mL with distilled water；Carbonate delays
The formula rushing liquid is: Na2CO30.105g、NaHCO30.756g, it is settled to 1000mL, pH8.0 with distilled water.
Choosing oil degradation bacteria new strains B11, be inoculated in fluid medium, controlling initial inoculation concentration is
1O7Cells/mL, in constant temperature air bath shaking table 30 DEG C, 180rpm cultivates 5 days.Draw inoculum after five days and carry out biological table
Face activating agent produces aptitude tests experiment.Concretely comprise the following steps:
On microtitration plate plate lid, drip the crude oil of 2 μ L, and at room temperature place 1 hour, backward in the middle of add 5 μ L
Inoculum.Wait 1 minute under room temperature condition, observe the shape of inoculum drop.Found that inoculum
Open shape in tiling and area is bigger.It follows that this antibacterial has the good ability producing biosurfactant.
Embodiment 4
Di Monei Marseille bacterium J42 emulsibility
Investigate cultivation temperature, initial pH value of medium and three factors of the bacteria concentration shadow to antibacterial emulsifying activity size respectively
Ring.
Liquid culture culture medium prescription used by the present embodiment is: carbonate buffer solution 1000mL, MgSO4·7H2O
0.1g、NH4Cl 0.5g、Na2HPO40.5g、CaCl2·6H2O 0.01g、FeSO4.7H2O0.005g, toluene 5g, trace element
1mL；Wherein, the formula of trace element is: ZnSO4·7H2O 0.44g、CuSO4·5H2O 0.20g、MnSO4·2H2O
0.17g、Na2MoO4·2H2O 0.06g、H3BO30.10g、CoCl2·6H2O 0.08g, it is settled to 1000mL with distilled water；Carbon
The formula of phthalate buffer is: Na2CO30.105g、NaHCO30.756g, it is settled to 1000mL, pH 8.0 with distilled water.
1) cultivation temperature impact on antibacterial emulsifying activity
The initial inoculation concentration accessing embodiment 1 acquisition is 107The bacterial strain J42 of cells/ml, is 6 and shaking speed at pH value
For 180rpm, cultivation temperature gradient is liquid culture 5 days under conditions of 25 DEG C, 30 DEG C and 35 DEG C.Vibrate after completing antibacterial culturing
Mixing inoculum, takes central liquid and carries out emulsification experiment, concretely comprise the following steps:
Clean each hexadecane adding 1ml in the cuvette dried to 3 respectively, be separately added into the thin of correspondence the most again
The each 1ml of bacteria culture fluid.Concussion mixes 2min and stands 10min the most at a high speed, observes respectively and records emulsion layer
Thickness and liquid gross thickness, and the emulsifying activity size at different temperatures of oil degradation bacteria is represented with their ratio.
Result display J42 bacterium when cultivation temperature is 25 DEG C, 30 DEG C and 35 DEG C, its emulsifying activity is respectively 0%,
47.76% and 15.82%, when cultivation temperature is 30 DEG C, emulsifying activity effect is best, antibacterial under the conditions of the higher temperature of gentleness on the low side
Emulsifying activity is all suppressed.
2) initial pH value of medium impact on antibacterial emulsifying activity
The initial inoculation concentration accessing embodiment 1 acquisition is 107The bacterial strain J42 of cells/ml, turns in temperature 30 DEG C and shaking table
Speed 180rpm, initial pH value of medium gradient is liquid culture 5 days under conditions of 6,7,8.Complete vibration mixing after antibacterial culturing
Inoculum, takes central liquid and carries out emulsification experiment, concretely comprise the following steps:
Each hexadecane adding 1ml and corresponding inoculum in the cuvette dried is cleaned respectively each to 3
1ml.At a high speed concussion mixing 2min also stands 10min, observe and respectively record emulsion layer thickness and liquid gross thickness and they
Ratio.
Result display J42 bacterium is when pH value is 6,7 and 8, and its emulsifying activity is respectively 47.76%, 57.48% and
50.25%, surface J42 bacterium emulsifying effectiveness when pH value is 7 is best.
3) bacteria concentration impact on antibacterial emulsifying activity size
The initial inoculation concentration accessing embodiment 1 acquisition is 107The bacterial strain J42 of cells/ml, temperature 30 DEG C and shake
Liquid culture 5 days under conditions of bed rotating speed 180rpm.It is centrifuged 15min under the conditions of 10000rpm after completing antibacterial culturing, separates thin
Bacterium thalline and supernatant.Preparing bacteria suspension with sterilized water dilution microorganism, controlling bacteria concentration after antibacterial dilutes is 106cells/
ml、107Cells/ml and 108Cells/ml, carries out emulsification experiment, concretely comprises the following steps:
Result shows that J42 bacterium is 10 at bacterial concentration6cells/ml、1O7Cells/ml and 108During cells/ml, its emulsifying
Activity is respectively 0%, 10.00% and 47.76%, and the highest emulsifying activity of bacteria concentration is the strongest.
It follows that 30 DEG C of cultivation temperature, the initial pH of culture medium be 7 and bacterium dense the biggest in the case of, J42 bacterium has good
Good emulsifying activity.
Embodiment 5
Investigate cultivation temperature and initial pH value of medium impact capillary on inoculum respectively.
Liquid culture culture medium prescription used by the present embodiment is: carbonate buffer solution 1000mL, MgSO4·7H2O
0.1g、NH4Cl 0.5g、Na2HPO40.5g、CaCl2·6H2O 0.01g、FeSO4.7H2O0.005g, toluene 5g, trace element
1mL；Wherein, the formula of trace element is: ZnSO4·7H2O 0.44g、CuSO4·5H2O 0.20g、MnSO4·2H2O
0.17g、Na2MoO4·2H2O 0.06g、H3BO30.10g、CoCl2·6H2O 0.08g, it is settled to 1000mL with distilled water；Carbon
The formula of phthalate buffer is: Na2CO30.105g、NaHCO30.756g, it is settled to 1000mL, pH8.0 with distilled water.
Before the experiment of bacterium surface tension force starts, first by the surface tension of full automatic watch/interfacial tensimeter determination experiment material,
Concretely comprise the following steps:
First open power switch device and analyzer storm door, take off adopting platinum plate with tweezers and link up with burning on alcohol burner flame envelope
Remove the impurity above adopting platinum plate, finally adopting platinum plate is hung back on instrument hook.It is washed with deionized water clean culture dish again, after drying
Pouring fluid medium into makes liquid level be 3-7mm, is positioned over immediately below adopting platinum plate on platform.Then shut storm door, treat white
Gold plate hook stablize motionless after press peeling key, press " upwards " again after data display screen display numerical stability, wait reality to appear
Test result.Press " downwards " after reading experimental result, wait adopting platinum plate to terminate experiment by " stopping " after departing from liquid.Repeated measure 3 times,
Average the surface tension as fluid medium.
Test result indicate that culture medium adding before toluene, surface tension is 72.16mN/m.
1) cultivation temperature impact on antibacterial emulsifying activity size
The initial inoculation concentration accessing embodiment 1 acquisition is 107The bacterial strain J42 of cells/ml, is 6 and shaking table at pH value
Rotating speed is 180rpm, and cultivation temperature gradient is liquid culture 5 days under conditions of 25 DEG C, 30 DEG C and 35 DEG C.After completing antibacterial culturing,
Centrifugal 15min, separation of bacterial thalline and supernatant under the conditions of 10000rpm.Collect centrifuged supernatant to survey for surface tension
Fixed.
Experimental result display J42 bacterium when cultivation temperature is 25 DEG C, 30 DEG C and 35 DEG C, the surface of culture fluid after cultivating five days
Tension force is respectively 34.86mN/m, 53.35mN/m and 46.71mN/m.Result shows when cultivation temperature is 25 DEG C reducing liquid table
Surface tension has obvious effect.
2) surface tension of inoculum under the conditions of culture medium difference original ph
The initial inoculation concentration accessing embodiment 1 acquisition is 107The bacterial strain J42 of cells/ml, temperature 30 DEG C and shake
Bed rotating speed 180rpm, initial pH value of medium gradient is liquid culture 5 days under conditions of 6,7,8.After completing antibacterial culturing,
Centrifugal 15min, separation of bacterial thalline and supernatant under the conditions of 10000rpm.Collect centrifuged supernatant, survey for surface tension
Fixed.
Experimental result display J42 bacterium is when initial pH value of medium is 6,7 and 8, and after cultivating five days, the surface of culture fluid is opened
Power is respectively 53.35mN/m, 54.18mN/m and 54.39mN/m.Surface J42 bacterium has the capillary ability of certain reduction.
It can thus be appreciated that J42 bacterial strain has the effect significantly reducing surface tension of liquid under 25 DEG C and specified conditions.
Embodiment 6
The degraded of toluene momomers is applied by new strains J42
The LB culture medium prescription of the present embodiment is: yeast extract 5.0g, peptone 10.0g, sodium chloride 10.0g, fine jade
Fat 15.0g, pH 7.2-7.4.
Liquid culture culture medium prescription used by the present embodiment is: carbonate buffer solution 1000mL, MgSO4·7H2O
0.1g、NH4Cl 0.5g、Na2HPO40.5g、CaCl2·6H2O 0.01g、FeSO4.7H2O0.005g, toluene 5g, trace element
1mL；Wherein, the formula of trace element is: ZnSO4·7H2O 0.44g、CuSO4·5H2O 0.20g、MnSO4·2H2O0.17g、
Na2MoO4·2H2O 0.06g、H3BO30.10g、CoCl2·6H2O 0.08g, it is settled to 1000mL with distilled water；Carbonate delays
The formula rushing liquid is: Na2CO30.105g、NaHCO30.756g, it is settled to 1000mL, pH 8.0 with distilled water.
Taking a certain amount of analytically pure toluene reagent, be dissolved in analytical pure methanol, being configured to concentration respectively is 10,20,30,
The standard solution of the toluene of 40,50,60 μ g/ml, is used for testing after the filtering with microporous membrane with 0.45 μm aperture.Use efficient liquid phase
Chromatography carries out the mensuration of toluene standard curve.
Experimental result display toluene retention time in the liquid-phase chromatographic column of C18 is 9.18min.Sit with peak area for vertical
Mark, benzene concentration are abscissa, and the standard curve of toluene is as it is shown in figure 1, obtain toluene normal equation and be: y=-8.388+
2.717x。
The mono-colony inoculation of the J42 domestication of above-mentioned toluene momomers preserved obtains being enriched with bacterium after activation 48h in LB culture medium
Liquid, is used for carrying out the degradation experiment of toluene momomers.
Experiment there is certain volatility due to toluene, so sample need to be analyzed after sampling immediately, and once opens aluminum
Culture bottle cannot be put back to and continue in shaking table to cultivate by lid once again.So J42 bacterium arranges five repetitions, its condition of culture during experiment
It is: initial inoculation concentration 107Cells/ml, temperature 30 DEG C, pH value is 6, shaking speed 180rpm.Cultivate the 2nd, 4,6,8,10
It time respectively open a bottle and carry out high performance liquid chromatography, the content of toluene in the middle of detection.1 blank assay is being set simultaneously,
I.e. condition of culture is consistent with above-mentioned cultivation, the most not inoculated bacteria.When placing 1,10 days in shaking table, in the middle of detection, toluene contains
Amount.
Toluene level when data when the 0th day come from blank the 1st day in Fig. 2.Known that J42 bacterium processes first by Fig. 2
During benzene aquatic solution, when the 8th day, in water, inspection did not measured toluene level.Figure shows the speed of degraded toluene first quick and back slow, may
Reason is along with the bacteriogenic biosurfactant of the growth metabolism of antibacterial is more and more, emulsifying activity is stronger,
Cause toluene to strengthen at the dissolubility of aqueous solution, and along with the reduction of toluene level, the disulfonic gas of volatilization also begins to again return
In the middle of solution, thus fallen by B11 bacterial degradation.Test result indicate that J42 bacterium reaches 100% to the degraded of toluene in solution.
Embodiment 7
The embodiment of the present invention additionally provides above-mentioned new strains and applies the degraded of COD in petroleum wastewater, as follows:
The J42 bacterium tamed by above-mentioned toluene momomers is with 107The inoculum concentration of cells/ml be inoculated into 100ml 10% crude oil without
In machine salt culture medium, temperature 30 DEG C, cultivate 7 days under conditions of shaking speed 180rpm.
Described 10% crude oil minimal medium: i.e. add 100ml crude oil in the minimal medium of 900ml system.
Described minimal medium includes: carbonate buffer solution 1000mL, MgSO4·7H2O 0.1g、NH4Cl 0.5g、
Na2HPO40.5g、CaCl2·6H2O 0.01g、FeSO4.7H2O 0.005g, trace element 1mL.
Described carbonate buffer solution includes: Na2CO30.105g、NaHCO30.756g, pH8.0 and distilled water 1000mL.
Described trace element includes: ZnSO4·7H2O 0.44g、CuSO4·5H2O 0.20g、MnSO4·2H2O0.17g、
Na2MoO4·2H2O 0.06g、H3BO30.10g、CoCl2·6H2O 0.08g, distilled water 1000mL.
Every other day draw 1.5ml bacteria suspension 12000rpm in the EP pipe of 1.5ml from each bottle and be centrifuged Aspirate supernatant 1ml
In 10ml color comparison tube, it is separately added into 9ml distilled water diluting.First weigh 0.12g HgSO4In digestion tube, add 50ul H2SO4
With 450ul H2O shakes to clearing up.Add 6ml H2SO4With Ag2SO4Mixed solution, adds 1ml K2CrO4Solution, adds
3ml sample (blank tube add 3ml distilled water) mixing, is placed in and clears up 165 DEG C of instrument and clear up 15 minutes.2 are cooled down again in clearing up instrument
Minute take out continue cooling after, add 3ml H2O, mixing, measure COD value to precipitation (in pipe, solution is without obvious floating particle).
Cuvette rinse adds solution in blank tube after drying, and COD measuring instrument display screen clicks on blank key, then clicks on measurement key, now
COD value is shown as 0.It is sequentially added into each sample in cuvette, measures COD value.
Measurement result shows that testing J42 bacterium is reduced to by 6030mg/L the 7th day COD value when Refinery Wastewater
3290mg/L, COD clearance is at the 7th day close to 50%, and COD degradation rate has and improves possibility further.
By above example, show that new strains J42 has good biosurfactant production ability, emulsifying activity, fall
Low surface tension of liquid ability and petroleum degradation rate are high.
Claims (10)
1. a strain Di Monei Marseille bacterium new strains, named J42.
2. as described in right 1, it is characterised in that described new strains has preferable biosurfactant production ability, emulsifying power
Power, reduction surface tension of liquid ability.
3. the purposes in degraded toluene of the Di Monei Marseille bacterium new strains described in claim 1.
Apply the most as claimed in claim 3, it is characterised in that during experiment, J42 bacterium arranges five repetitions, in same culture conditions
Under take out every three days a culture bottle carry out high-efficient liquid spectrum detection solution in toluene level.
Apply the most as claimed in claim 4, it is characterised in that described J42 bacterium condition of culture is for being placed on 30 DEG C of constant-temperature tables
180rpm, cultivates 10 days.
Apply the most as claimed in claim 4, it is characterised in that before cultivating, the inoculum density of thalline is 107cells/ml。
7. the purposes in degraded oil waste water of the Di Monei Marseille bacterium new strains described in claim 1.
8. the purposes as described in right 7, it is characterised in that in degradation experiment J42 bacterium access be concentration be 10% crude oil inorganic
Salt culture medium.
9. the purposes as described in right 8, it is characterised in that: 100mL crude oil adds 900mL inorganic salt liquid culture medium.
10. the purposes as described in right 9, it is characterised in that inorganic salt liquid culture medium contains: carbonate buffer solution 1000mL,
MgSO4·7H2O0.1g、NH4Cl0.5g、Na2HPO40.5g、CaCl2·6H2O0.01g、FeSO4.7H2O0.005g, trace element
1mL；Described carbonate buffer solution includes: Na2CO30.105g、NaHCO30.756g, pH8.0 and distilled water 1000mL；Described micro-
Secondary element includes: ZnSO4·7H2O0.44g、CuSO4·5H2O0.20g、MnSO4·2H2O0.17g、Na2MoO4·
2H2O0.06g、H3BO30.10g、CoCl2·6H2O0.08g, distilled water 1000mL.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471598A (en) * | 2020-04-25 | 2020-07-31 | 甘肃省科学院生物研究所 | Gliocladium roseum and pimavalia rimonaris composite microbial agent and application thereof in prevention and control of diseases and pests |
| CN111500494A (en) * | 2020-04-25 | 2020-08-07 | 甘肃省科学院生物研究所 | Thymus damascena with high enzyme activity and screening method and application thereof |
| CN113893266A (en) * | 2021-12-10 | 2022-01-07 | 达尔文新研(北京)生物科技有限公司 | Composite mosaic bacterium extract, preparation method and application thereof |
| CN114958664A (en) * | 2022-05-24 | 2022-08-30 | 昆明学院 | Malelia strain KC009, fermentation broth thereof and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111471598A (en) * | 2020-04-25 | 2020-07-31 | 甘肃省科学院生物研究所 | Gliocladium roseum and pimavalia rimonaris composite microbial agent and application thereof in prevention and control of diseases and pests |
| CN111500494A (en) * | 2020-04-25 | 2020-08-07 | 甘肃省科学院生物研究所 | Thymus damascena with high enzyme activity and screening method and application thereof |
| CN113893266A (en) * | 2021-12-10 | 2022-01-07 | 达尔文新研(北京)生物科技有限公司 | Composite mosaic bacterium extract, preparation method and application thereof |
| CN113893266B (en) * | 2021-12-10 | 2022-03-25 | 达尔文新研(北京)生物科技有限公司 | Composite mosaic bacterium extract, preparation method and application thereof |
| CN114958664A (en) * | 2022-05-24 | 2022-08-30 | 昆明学院 | Malelia strain KC009, fermentation broth thereof and application thereof |
| CN114958664B (en) * | 2022-05-24 | 2023-06-02 | 昆明学院 | Maxi Li Yajun strain KC009, fermentation liquor and application thereof |
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