CN108002548A - One plant of method with the efficient pyrene degradation function bacterial strain BW02 efficient degradation polycyclic aromatic hydrocarbons of low temperature salt tolerant - Google Patents
One plant of method with the efficient pyrene degradation function bacterial strain BW02 efficient degradation polycyclic aromatic hydrocarbons of low temperature salt tolerant Download PDFInfo
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Abstract
This divisional application is related to one plant of method with the efficient pyrene degradation function bacterial strain BW02 efficient degradation polycyclic aromatic hydrocarbons of low temperature salt tolerant.The bacterial strain deposit number is CGMCC No.10354, its 16SrRNA sequence such as SEQ ID No:Shown in 1.The bacterial strain BW02 that the present invention screens has the function of efficient degrading polycyclic aromatic hydrocarbons, the bacterial strain has good degradation property under the conditions of neutral and meta-alkalescence, it is relatively low for oxygen demand, pyrene degradation rate is high when 15 DEG C of low temperature environments, salinity 3%, it is adapted to environments such as subsea, there is good degradation for the polycyclic aromatic hydrocarbon in oceanographic sedimentation pollutant.
Description
The application is Application No. 2017111277435, the applying date is on November 15th, 2017, " one plant entitled
With the efficient pyrene degradation function bacterial strain BW02 of low temperature salt tolerant and its purposes " divisional application.
Technical field
The invention belongs to microbial technology field, and in particular to one plant has the efficient pyrene degradation function bacterial strain of low temperature salt tolerant
BW02 and its purposes.
Background technology
Polycyclic aromatic hydrocarbon (Polycyclic Aromatic Hydrocarbons abbreviation PAHs)：Refer to 2 and more than 2 benzene
Ring be combined with each other, and the fused ring compound formed with the arrangement of wire, horn shape or tufted, base unit is phenyl ring, and stable structure is not easy
Degraded, and by the production of biological accumulation and biological magnification to the mankind and life serious health can be caused to endanger
Evil.Containing 2-3 phenyl ring, be referred to as low molecular weight PAHs (Low-molecular-weight, abbreviation LMW-PAHs), as naphthalene,
Anthracene and phenanthrene.High molecular weight PAHs (High-molecular-weight, abbreviation HMW-PAHs) with 4-6 phenyl ring, as pyrene,
Benzo R and benzo [a] pyrene etc..Pyrene is made of four phenyl ring, is the representative thing of high ring polycyclic aromatic hydrocarbon, itself does not produce cell
Toxicity, intake human body form class compound of waking up by being metabolized, such material has strong carcinogenicity.In general, high molecular weight
PAHs steady chemical structures, it is difficult to utilized by general Institute of Micro-biology.
In recent years, drastically accelerate as the natural resources exploitations such as the fast development of industry, oil, coal, natural gas utilize,
Polycyclic aromatic hydrocarbons contaminated in environment has obvious ascendant trend.Hitherto it is found that more than 200 kinds of polycyclic arene compound.Wherein
With containing substantial amounts of polycyclic aromatic hydrocarbon in air in marine sediment, terrestrial soil, because of its stability with height, difficult drop
Solution, strong toxicity, and the extensive concern that there is the harm such as cumulative effect to be subject to environmental science person, many countries and regions are more cyclophanes
Hydrocarbons are classified as one of serious environmental contaminants, U.S.EPA be even more in the early 1980s by 16 plants not
PAHs with branch is determined as priority pollutants in environment.In nature (soil pollutant, oceanographic sedimentation pollutant)
There are many microorganisms for having degradation to polycyclic aromatic hydrocarbon, these microorganisms have it is widely distributed, metabolic type is various, degraded
Power efficient, metabolite are various, environment adapts to the advantages that stability is high compared with strong and genetic variability, thus become postgraduate
The hot spot of thing degraded.But different microbial degradation abilities is different, and degradation mechanism is also different, therefore find optimum degraded
The bacterial strain of pyrene causes the concern of more and more researchers.
The content of the invention
The present invention is to make up the deficiencies in the prior art, there is provided one plant has the efficient pyrene degradation function bacterial strain of low temperature salt tolerant
BW02 and its purposes.
One plant has low temperature salt tolerant efficient pyrene degradation function bacterial strain BW02, Latin name Rhodococcus
qingshengii.Its 16SrRNA sequence such as SEQ ID No:Shown in 1.The bacterial strain BW02 is collected in Talien New Port petroleum wastewater
Contaminate marine site, concentration and separation gained.The psychrotropic bacteria BW02 with pyrene degradation function has been filed on preservation, specific preservation information
It is as follows：
Depositary institution's title：China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC)；
Depositary institution address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica；
Preservation date：On 01 13rd, 2015；
Deposit number：CGMCC No.10354；
Applications of the above-mentioned bacterial strains BW02 on efficient degradation polycyclic aromatic hydrocarbon pyrene is claimed in second purpose of the invention.
The method that above-mentioned bacterial strains BW02 degraded pyrenes are claimed in 3rd purpose of the invention, step are：
1) ASM culture mediums are added to conical flask, in 7.5,121 DEG C of sterilizing 30min of pH；
2) trace element solution 0.5ml and pyrene are separately added into the ASM culture mediums for bacterium of having gone out, make the pyrene ultimate density be
1mg/ml；
3) choose the bacterial strain that cell age is 48h and prepare bacteria suspension, control the cell concentration 10 of bacterial strain9Cfu/ml, connects with 8%
Kind amount access pyrene content is in the ASM culture mediums of 1mg/ml, and simulated sea bottom environment anaerobic condition, adjusts medium pH 7.5, in 15
DEG C quiescent culture 15d.
Further, the ASM culture medium prescriptions：
NaCl：30g, NH4NO3：1g, KH2PO4：0.2245g, Na2HPO4·12H2O：5g, natural sea-water 1000ml, it is micro
Element Solution：10ml.
Further, the trace element solution formula：CaCl22mg, FeCl6H2O 50mg, CuSO40.5mg,
MnCl·4H2O 0.5mg, ZnSO4·7H2O 10mg, distilled water 1000ml.
The bacterial strain BW02 that the present invention screens has efficient pyrene degradation function, which has under the conditions of neutral and meta-alkalescence
Good degradation property, relatively low for oxygen demand, pyrene degradation rate is high when 15 DEG C of low temperature environments, salinity 3%, fits
Environments such as subsea is closed, there is good degradation for the polycyclic aromatic hydrocarbon in oceanographic sedimentation pollutant.
Brief description of the drawings
Fig. 1 bacterial strain BW02 colonial morphologies；
Fig. 2 bacterial strain BW02 electron-microscope scanning forms；
The structure of the phylogenetic tree of Fig. 3 bacterial strains BW02；
The growth curve of Fig. 4 bacterial strains BW02；
Influence of Fig. 5 cell ages to bacterial strain BW02 pyrene degradation properties；
Influence of Fig. 6 inoculum concentrations to bacterial strain BW02 pyrene degradation properties；
Fig. 7 difference pH, temperature and influence of the rotating speed to bacterial strain BW02 pyrene degradation properties；
Influence of Fig. 8 NaCl concentrations to bacterial strain BW02 pyrene degradation characteristics；
Influence of Fig. 9 times to bacterial strain BW02 pyrene degradation properties；
The inorganic nitrogen-sourced influences to bacterial strain BW02 pyrene degradation properties of Figure 10；
Figure 11 Na2HPO4Influence of the concentration to bacterial strain BW02 pyrene degradation properties；
Figure 12 bacterial strains BW02 in the degradation process of pyrene to producing metabolite.
Embodiment
The present invention is described in detail below by the drawings and specific embodiments, but is not limited the scope of the invention.Such as without special
Illustrate, experimental method of the present invention is conventional method, and experiment equipment used, material, reagent etc. can be from business ways
Footpath obtains.
Embodiment 1
Bacterial strain BW02 is cultivated
LB culture mediums (g/L):Tryptone (Tryptone) 10g；Agar (Agar) 15g；Yeast extract (Yeast
Extract) 5g, NaCl 10g, deionized water 1L, adjust pH to 7.0, fluid nutrient medium does not add agar then.
ASM inorganic salts culture (g/L):NaCl 30g；MgSO4, 0.35g；NH4NO31g；KCI 0.7g,
Na2HPO4.12H2O 5g；KH2PO40.2245g；PH7.5, finally adds 100ul trace element solutions.
Trace element solution (mg/L):CaCl22mg；FeCl3.6H2O5mg；GuSO40.5mg；MnCl2.4H20 0.5mg；
ZnSO4.7H2O 10mg；Distilled water 1000ml.
Its colonial morphology is observed after 48h will be cultivated in inoculation after purification to LB solid mediums.At the same time will be appropriate
Concentration strain to be tested bacteria suspension drop cures 2-4h with 2.5% glutaraldehyde, then uses pH=7.3 on the silicon chip of 5mm × 4mm
Phosphate buffer washing, Gradient elution using ethanol, finally using isoamyl acetate into line replacement, vacuum drying, uses scanning electron
Microscope (Hitachi's S-4800 Flied emissions) carries out thalline observation.
(1) colony morphology characteristic
The colonial morphology of bacterial strain BW02 is：After being cultivated 7 days on LB culture mediums, bacterium colony circular protrusions, yellow pink, surface light
Sliding moistening, neat in edge.
(2) strain morphology and its physio-biochemical characteristics
It is in crocus on LB solid mediums as shown in Figure 1, efficient pyrene degradation bacteria strains BW02 Gram-positives, surface
Smooth bumps are moistened, neat in edge is opaque.As shown in Fig. 2, thalline is rod-shaped, non-paired appearance, atrichia, is about 0.8-2
μm, it is about 0.3-0.6 μm wide.Bacterial strain BW02 can be using sucrose, lactose, dextrin as sole carbon source, it is impossible to utilizes trehalose, inulin and sweet
Reveal alcohol.(G+C) mol% is 59.1%.It can be grown in the range of NaCl concentration 0-20%, most suitable NaCl concentration 3%, growth
4-30 DEG C of temperature range, 15-20 DEG C of optimum temperature range, pH scope 4.5-10.5, optimal pH 7.0-8.0,
Utilize 16SrRNA sequence construct bacterial strain BW02 phylogenetic trees (Fig. 3), bacterial strain BW02 and Rhodococcus
Qingshengii and Rhodococcus degradans affiliations are nearest, and homology is up to 98.03%, with reference to the shape of bacterial strain
State feature and analysis of physio biochemical characteristics, the identified R.qingshengii of bacterial strain BW02.
Embodiment 2
The measure of the growth curve of efficient pyrene degradation bacteria strains BW02
Experiment using visible spectrophotometry value method measure pyrene efficient degrading bacterial strain growth curve, Detection wavelength 600nm,
Using sterile fluid nutrient medium as blank control, it is right in 6,12,18,24,36,42,54,66,72,78,84h institutes to detect respectively
The OD answered600.Three times, be averaged (if bacteria suspension concentration is too dense, should do appropriate dilution, make OD every part of sample measure600Value is less than
1)。
Under the suitable conditions, thalli growth occurs 4 stages to bacterial strain BW02, and lag phase, logarithmic phase, stationary phase, decline
Die the phase.Test result indicates that：0-20h is lag phase, and thalli growth is slow；20-60h is logarithmic phase, and thalline mushrooms out；60-
90h is stationary phase, and thalli growth gradually tends to be steady.Such as Fig. 4.
Embodiment 3
Influence of the cell age to bacterial strain BW02 pyrene degradation properties
Have chosen cell age respectively according to growth curve is 24h, 48h, 72h, and the bacterial strain of 96h prepares bacterium as research object
The bacteria suspension of strain BW02, adjusts cell concentration cfu/ml=109.Pyrene content is respectively connected to as 1mg/ml's using 4% inoculum concentration
In 50mlASM fluid nutrient mediums, adjust pH7.5,15 DEG C of quiescent culture 15d, each sample do 3 it is parallel, not connect bacterium to be empty
White control, influence of the different cell ages to bacterial strain BW02 pyrene degradation properties is measured using gas chromatography.
The cell age of bacterial strain BW02 (Rhod) different times, respectively 24h, 48h, 72h, the bacterial strain of 96h are chosen in experiment
Analyzed, degradation property is optimal when cell age as shown in Figure 5 is 48h, up to more than 60%.
Embodiment 4
Influence of the inoculum concentration to bacterial strain BW02 pyrene degradation properties
The bacteria suspension of bacterial strain BW02 is prepared, adjusts cell concentration cfu/ml=109.Respectively with 1%, 2%, 4%, 6%,
8%, 12% inoculum concentration is respectively connected in the 50mlASM fluid nutrient mediums that pyrene content is 1mg/ml, adjusts pH7.5,15 DEG C quiet
Put culture 15d, each sample do 3 it is parallel, not connect bacterium as blank control, using gas chromatography measure different vaccination amount pair
The influence of bacterial strain BW02 pyrene degradation properties.
When 1%-4%, degradation rate substantially increases rapidly inoculum concentration；When inoculum concentration is 4%-8%, the degradation rate of pyrene delays
Slow increase, when inoculum concentration reaches 8%, the degradation rate of pyrene reaches highest, is hereafter stepped up with inoculum concentration, and degradation rate is not
It may proceed to increase, gradually tend to be steady.
Embodiment 5
PH, temperature, rotating speed, influence of the salinity to bacterial strain BW02 pyrene degradation properties
The bacteria suspension of bacterial strain BW02 is prepared, adjusts cell concentration cfu/ml=109.Pyrene is respectively connected to 4% inoculum concentration
Content is in the 50mlASM fluid nutrient mediums of 1mg/ml, and it is respectively 6.0,6.5,7.0,7.5,8.0,15 DEG C of standing trainings to adjust pH
Support 15d, each sample do 3 it is parallel, not connect bacterium as blank control, different pH value are measured to bacterial strain using gas chromatography
The influence of BW02 pyrene degradation properties；In addition identical method is used, when pH is respectively 7.5, temperature is respectively 10 DEG C, 15 DEG C,
At 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, influence of the measure different temperatures to bacterial strain BW02 pyrene degradation properties；At 15 DEG C, pH7.5, adjust
Section rotating speed is respectively 50rpm/min, 100rpm/min, 150rpm/min, 200rpm/min, and shaken cultivation 15d, measure is different to be turned
Influence of the speed to bacterial strain BW02 pyrene degradation properties.
Compare different pH value, temperature, influence of the rotating speed to bacterial strain BW02 pyrenes degraded main performance, such as Fig. 7, is 7.5 in pH
When degradation effect it is optimal (Fig. 7 A)；At 15 DEG C, bacterial strain BW02 is optimal (Fig. 7 B) to the degradation effect of pyrene；As rotating speed improves, bacterium
Strain BW02 reduces the degradation rate of pyrene on the contrary, illustrates that bacterial strain BW02 is relatively low (Fig. 7 C) to the demand of oxygen.As shown in Figure 8
For bacterial strain BW02 in NaCl concentration 3%, its pyrene degrading activity highest, reaches 61%.
Embodiment 6
Influence of the time to bacterial strain BW02 pyrene degradation properties
The bacteria suspension of excellent bacterial strain BW02 is prepared, adjusts cell concentration cfu/ml=109.8% inoculum concentration is respectively connected to
Pyrene content is 1mg | in the 50mlASM fluid nutrient mediums of ml, adjust pH7.5,15 DEG C of quiescent cultures, respectively at 4d, 6d, 8d,
10d, 16d and 20d are sampled, each sample do 3 it is parallel, not connect bacterium as blank control, measured using gas chromatography different
Influences of the period bacterial strain BW02 to pyrene degradation property.During 0-4d as shown in Figure 9, degradation rate increases sharply, degradation rate after 8d
Increase is relatively slow, and 16d degradation rates reach maximum, and degradation rate tends to be steady thereafter.
Embodiment 7
The different inorganic nitrogen-sourced influences to bacterial strain BW02 pyrene degradation properties
Three kinds of different inorganic nitrogen-sourced NH are chosen in experiment4Cl, NaNO3, NH4NO3, trained according to the optimal conditions completed above
Support, measure the different inorganic nitrogen-sourced influences to bacterial strain BW02 pyrene degradation properties.
Using gas chromatography, the inorganic nitrogen-sourced NH of analysis addition 0.5g/L4Cl, NaNO3, NH4NO3Bacterial strain pyrene is dropped
Solve characteristic influence the result shows that, add NH4NO3Culture medium reach 49% most beneficial for degraded of the bacterial strain to pyrene, degradation rate
(Figure 10).
Embodiment 8
Influence of the phosphorus source of various concentrations to bacterial strain BW02 pyrene degradation properties
Culture medium is prepared according to the condition of culture above optimized, it is 1mg/ that 8% inoculum concentration, which is respectively connected to pyrene content,
In the 50mlASM fluid nutrient mediums of ml, wherein adjusting Na2HPO4Concentration is respectively 1g/L, 3g/L, 5g/L, 7g/L, 9g/L.Adjust
After pH7.5,15 DEG C of quiescent culture 16d, using the Na of gas chromatography analysis various concentrations2HPO4Degrade to bacterial strain BW02 pyrenes
The influence of performance.
PO3-It is one of necessary ion of thalli growth, PO under normal conditions3-Reason suffers from thalli growth important
Effect, as seen from Figure 11, works as Na2HPO4When concentration is 3g/L, bacterial strain BW02 reaches the degradation rate highest of pyrene, degradation rate
55.23%.
Embodiment 9
The measure of bacterial strain BW02 pyrenes degraded mesostate
Using GC-MS methods, with 0.22um organic solvent-resistant membrane filtration petroleum ether extraction liquid.Filtrate is suitably diluted,
GC-MS analyses are carried out, to measure the serial mesostate of pyrene.GC-MS conditions：Injection port takes shunt mode, split ratio
For 20：1, nonpolar gas chromatographic column (Rxi-5Sil, HP-5).Injector temperature is 300 DEG C, and FID temperature is 250 DEG C, program
Heat up initial column temperature be 100 DEG C, keep 2min, then with 8 DEG C/min temperature programmings to 220 DEG C, keep 10min, then with 6 DEG C/
Min temperature programmings keep 10min to 280 DEG C.
Analyzed by GC-MS, detect bacterial strain BW02 to producing a series of metabolites, parent in the degradation process of pyrene
Compound (pyrene) elution time is 20.472min, the first metabolite appears in 20.090min, base peak in 218 (100%), with
Spectrum storehouse compares, and it is 1-Hydroxy prene to determine this kind of material.Second of metabolin occurs in 20.113min, base peak (m/
Z) in 208 (100%), relatively determine that the material is 1-Methoxy phenanthrene. gas phases compared with the data inside mass spectrum
The 3rd metabolite appears in 17.365min in chromatography-mass spectroscopy storehouse, and base peak compared with MS composes storehouse, is determined in 149 (100%)
For the material to appear in 27.644min shown in the 4th metabolite of Benzoic acid., base peak is in (100%), with MS storehouses
Standard Pentanoic acid coincide.
Embodiment 10
Bacterial strain BW02 pyrenes and degrading polycyclic aromatic hydrocarbons performance measurement
1) conical flask of 150ml adds 121 DEG C of sterilizing 30min of ASM culture mediums 50ml/ bottles；
ASM culture medium prescriptions：
NaCl：30g, NH4NO3：1g, KH2PO4：0.2245g, Na2HPO4·12H2O：5g, natural sea-water 1000ml, it is micro
Element Solution：10ml, pH 7.5.121 DEG C of 30min of sterilising temp.
Trace element solution formula：
| CaCl2 | FeCl.6H2O | CuSO4 | MnCl.4H2O | ZnSO4·7H2O | Distilled water |
| 2mg | 50mg | 0.5mg | 0.5mg | 10mg | 1000ml |
2) pyrene and other polycyclic aromatic hydrocarbons (phenanthrene, naphthalene, anthracene) will be separately added into the culture medium for bacterium of having gone out, makes the ultimate density be
1mg/ml and trace element solution 0.5ml；
3) choose the bacterial strain that cell age is 48h and prepare bacteria suspension, control the cell concentration 10 of bacterial strain9Cfu/ml, connects with 8%
Kind amount access pyrene content is in the ASM fluid nutrient mediums of 1mg/ml, adjusts pH7.5；
4) compareed with being not added with the blank ASM culture mediums (with the addition of 1mg/ml pyrenes) of bacterium；
5) in 15 DEG C of quiescent culture 15d, experiment is in triplicate；
6) pyrene and the assay method of other degrading polycyclic aromatic hydrocarbons rates：
After nutrient solution, extraction nutrient solution is carried out using petroleum ether in three times, constant volume is to 30ml, and by extract low temperature
It is sealed, in case gas chromatographic analysis.
Using the content of gas chromatography measure pyrene, instrument is the gas chromatograph of Shimadzu -2010, is equipped with FID detections
Device, Rts-1 capillary columns, determination condition are as follows：Injector temperature is 300 DEG C, and detector temperature is 300 DEG C, originates column temperature 50
℃.With 15 DEG C/min temperature programmings to 250 DEG C, 10min is kept；Carrier gas nitrogen, split sampling, split ratio 10:1, sample size
1ul.Gas chromatography quantitative approach：Not connect the ASM minimal mediums of bacterium as control, degradation rate D%=(CCK-Cf)/
CCK × 100%, wherein CCK are control treatment group pyrene concentration, and Cf is pyrene or other polycyclic aromatic hydrocarbon concentration after bacterium is handled.
Degrading polycyclic aromatic hydrocarbons situation as shown in table 1 understands that bacterial strain BW02 shows efficient degradation property for pyrene.
1 degrading polycyclic aromatic hydrocarbons situation of table
Claims (4)
1. one plant of method with the efficient pyrene degradation function bacterial strain BW02 efficient degradation polycyclic aromatic hydrocarbons of low temperature salt tolerant, its feature exist
In bacterial strain BW02 deposit numbers are CGMCCNo.10354, its degradation step is：
1) ASM culture mediums are added to conical flask, in pH7.5,121 DEG C of sterilizing 30min；
2) trace element solution 0.5ml and polycyclic aromatic hydrocarbon are separately added into the ASM culture mediums for bacterium of having gone out, make polycyclic aromatic hydrocarbon final
Concentration is 1mg/ml；
3) choose the bacterial strain that cell age is 48h and prepare bacteria suspension, control the cell concentration 10 of bacterial strain9Cfu/ml, with 8% inoculum concentration
Access in the ASM culture mediums that polycyclic aromatic hydrocarbon content is 1mg/ml, medium pH 7.5 is adjusted, in 15 DEG C of quiescent culture 15d.
2. according to the method described in claim 1, it is characterized in that, the polycyclic aromatic hydrocarbon is：One kind in naphthalene, phenanthrene, anthracene, pyrene or
It is a variety of.
3. the according to the method described in claim 1, it is characterized in that, ASM culture medium prescriptions：NaCl：30g, NH4NO3：1g,
KH2PO4：0.2245g, Na2HPO4·12H2O：5g, natural sea-water 1000ml, trace element solution：10ml.
4. the according to the method described in claim 1, it is characterized in that, trace element solution formula：CaCl22mg, FeCl
6H2O50mg, CuSO40.5mg, MnCl4H2O0.5mg, ZnSO4·7H2O10mg, distilled water 1000ml.
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| CN107815430A (en) | 2018-03-20 |
| CN107815430B (en) | 2020-05-08 |
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