CN107796906A - Rhodococcus sp degradation condition is improved based on metabolism group to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate - Google Patents
Rhodococcus sp degradation condition is improved based on metabolism group to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate Download PDFInfo
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Abstract
Rhodococcus sp degradation condition is improved to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate based on metabolism group the invention discloses a kind of, comprised the following steps：(1) metabolin of Rhodococcus sp intracellular is measured；(2) ability of Rhodococcus sp degraded pyrene is analyzed；(3) principal component (PCA) is analyzed；(4) process analysis procedure analysis；The present invention discloses the metabolin difference of Rhodococcus sp degrading polycyclic aromatic hydrocarbons pyrene using metabolism group means and combination difference significance analysis, further the explaination metabolic alterations mechanism related to polycyclic aromatic hydrocarbon pyrene degraded.By analyzing degraded pyrene ability of the Rhodococcus sp under different growing environment and metaboilic level, the metabolin related to degraded pyrene is found, so as to optimize its degradation condition, the raising for polycyclic aromatic hydrocarbon pyrene degradation rate provides direction.The optimizing research that this method is alternatively other degrading polycyclic aromatic hydrocarbons microbial Degradation Conditions provides new idea and method.
Description
Technical field
The invention belongs to microbial degradation pollutant field, and in particular to one kind improves Rhodococcus sp degraded based on metabolism group
Condition is to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate.
Background technology
With oil exploitation and the increase of usage amount, substantial amounts of oil and its processed goods enter environment, and crude oil gathers, is smart
Substantial amounts of polycyclic aromatic hydrocarbon can be produced during refining, transport, burning etc., inevitably environment is polluted, polycyclic aromatic hydrocarbon
Accumulation has threaten the health of the mankind increasingly severely, it has also become the organic pollution that countries in the world are paid close attention to jointly.It is polycyclic
Aromatic hydrocarbons refers to the hydrocarbon containing 2 or more than 2 phenyl ring in molecule, such as naphthalene, anthracene, phenanthrene, pyrene, biphenyl, terphenyl.It is more
PAH is widely present in soil, water body and air, poorly water-soluble, has heat endurance, and a wherein suitable part
All there is chronic toxicity and carcinogenic, teratogenesis, mutagenic " three cause " effect, huge danger is caused to human health and ecological environment
Evil is a kind of dangerous in environment and needs the compound of primary study.The phenanthrene of especially polycyclic class, pyrene (pyrene) etc. are extremely difficult in ring
Degraded in border, be a kind of environmental contaminants for needing improvement badly.
Compared with other processing methods, biological harnessing method have easy to operate, cost is cheap, non-secondary pollution, administer effect
Fruit is good and can recover the clear superiorities such as soil-grown ability.Microbial degradation plays an important role in biodegradation, degradable more
The microorganism of PAH includes bacterium, fungi and algae, and wherein bacterium plays main function in most of environment.Research at present
More is Rhodococcus sp, pseudomonad, and Rhod has good degraded to imitate for linear paraffin, branched paraffin and alkylbenzene
Fruit, and part Rhod also has the effect of desulfurization.Present invention selection has the red of preferable degradation effect to polycyclic aromatic hydrocarbon
Coccus illustrates.
Polycyclic aromatic hydrocarbon with high molecular weight such as pyrene, ring structure are difficult cracking, and its biodegradation is complex.Low molecule amount
Polycyclic aromatic hydrocarbon it is water-soluble preferably, can be degraded by multiple-microorganism；And for the more of HMW especially Fourth Ring and the above
PAH, its molecular structure are complicated, it is difficult to it be oxidized, and heat endurance is stronger, it is difficult to isolated applicable degradation bacteria, separation
There is also certain limitation for obtained degradation bacteria strains.Therefore present invention selection represents thing as polycyclic aromatic hydrocarbon using pyrene and carries out degraded bar
Part is improved and then improved to its degradation rate.
The low limitation of degradation efficiency be present for biodegradation, it can be it to improve to the biodegradable efficiency of polycyclic aromatic hydrocarbon pyrene
Its hard-degraded substance is biological prosthetic to be accelerated to provide reference and instruction.Nowadays biodegradable optimization method is directed to, it is use more
" black-box model ", the difference of degradation rate before and after degraded is focused simply on, lack for katabolism thing difference and recognize, metabolism substrate
Influence to cell metabolite is extremely complex.Metabolism group is that it is certainly by monitoring analysis cell by after environmental stimulus
The change of body metabolite or its change with time, be study biosystem change a science, it is focused mainly on
Relative molecular mass is less than 1000 endogenous small-molecule substance in organism.Substrate is degraded to centre based on metabonomic analysis
The difference of metabolin, culture medium can effectively be adjusted according to result, change cell metabolism feature, so as to improve the drop to object
Solution rate.
Rhodococcus sp degradation condition is improved to improve polycyclic aromatic hydrocarbon pyrene degradation rate based on metabolism group, it is necessary to change degraded bottom
Thing, so as to analyze the catabolic pathway of metabolism difference in microbial body, and the vital activity of microbial cell to a great extent by
To the influence of its life system surrounding enviroment, when a variety of toxic pollutant collective effects larger cell interior can be caused to be metabolized
Level change.Middle long chain alkane is more degradable compared to polycyclic aromatic hydrocarbon small toxicity, and has certain similarity with polycyclic aromatic hydrocarbon, all
For oil pollution organic matter.Therefore the analysis that other long chain alkanes carry out metabolin difference for carbon source is added in selection.Microorganism pair
In the metabolic process of pyrene, environment-stress may be caused because of the high toxicity of pyrene, microbial metabolism can be made to go out to protect the thing of cell
Matter.The control group of common long chain alkane is added, although microbial degradation is also more difficult, substrate toxicity is small to make it grow generation
Thank to more vigorous generation protein synthesis correlative metabolites.Analysis of key metabolin difference is just it is further known that pyrene is studied carefully as substrate
Unexpectedly microorganism which metabolic pathway is have impact on, and then targetedly adds nutriment or carries out strain improvement, is further improved
Degradation capability of the bacterial strain to polycyclic aromatic hydrocarbon pyrene.Key metabolites are generally the material for participating in microorganism central metabolism.
Rationality, the degradation rate of polycyclic aromatic hydrocarbon pyrene is effectively improved, it is necessary in system scope, effectively disclose intracellular organic matter
Metabolic mechanism, determine the limiting factor of degradation of substrates, the interactive relation between rational analysis metabolin, and then formulate rationality and change
Enter strategy, improve degradation condition, improve the biological degradation rate of target contaminant.
The content of the invention
The purpose of the present invention is overcome the deficiencies in the prior art, there is provided one kind improves Rhodococcus sp degraded bar based on metabolism group
Part is to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate.
Technical scheme is summarized as follows：
Rhodococcus sp degradation condition is improved based on metabolism group to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate, including following step
Suddenly：
(1) metabolin of Rhodococcus sp intracellular is measured；
(2) ability of Rhodococcus sp degraded pyrene is analyzed；
(3) principal component analysis (Principal Component Analysis (PCA))；
(4) process analysis procedure analysis.
The step (1) is measured method to the metabolin of Rhodococcus sp intracellular and is：Entered using different alkane and pyrene as substrate
Row degraded, degraded later stage are measured to the metabolin of Rhodococcus sp intracellular, collect and are quenched including cell, extract into the cell
Metabolin, metabolin derivatization, GC-MS detections.The methanol aqueous solution that liquid is 60% is quenched, dichloro is designated as in the metabolin of addition
Methane phenanthrene solution, derivatization reagent are the pyridine solution and N- methyl-N- (trimethyl silane) trifluoro second of methoxyl group ammonium salt hydrochlorate
Acid amides.
The ability of the pyrene of being degraded to Rhodococcus sp of the step (2) carries out analysis method and is：Rhodococcus sp under different substrates is dropped
Solution pyrene ability analyzed, including substrate after degraded is extracted, GC detect.The extract used is dichloromethane
Alkane.
Step (3) principal component analysis is to carry out principal component analysis, specific steps to metabolin in SIMCA13.0 softwares
Batch processing is carried out for the metabolin relative amount data of acquisition are imported into MZmine softwares, is carried out by SIMCA13.0 softwares
Mass Spectrometer Method, chromatographic peak structure, chromatographic peak is deconvoluted, base peak is alignd, peak is identified, final to obtain the initial qualitative, quantitative of metabolin
Data.
It is described as follows：
Step (1) includes cell and collects and be quenched, extracts endocellular metabolism thing, metabolin derivatization and GC-MS detections, tool
Gymnastics is made as follows：
1. cell is collected and is quenched：Need Rhodococcus sp being seeded to minimal medium culture before collecting cell；Culture medium
At least three groups of sole carbon sources added respectively are：Pyrene, pyrene+hypotoxicity alkane carbon source of other similar pyrenes, hypotoxicity alkane carbon
Source.
2. extract endocellular metabolism thing：Take 1. cell that step obtains in mortar plus liquid nitrogen grinding, ground thin
Extract solution and multigelation are added in born of the same parents；Freeze thawing liquid centrifuging and taking supernatant is deserved to be called into clear liquid a, lower confluent monolayer cells add extract solution from
The heart takes supernatant to deserve to be called clear liquid b, and supernatant a and b is well mixed that supernatant c, centrifuging and taking supernatant c go to centrifuge tube, added
Enter dichloromethane phenanthrene solution as internal standard, after mixing under the conditions of -40 DEG C, vacuum freeze drying obtains sample.The extract solution is body
Product concentration is 50% methanol aqueous solution；
3. metabolin derivatization：The pyridine solution of addition methoxyl group ammonium salt hydrochlorate in the sample 2. obtained to step, 40 DEG C
Water-bath 80min；N- methyl-N- (trimethyl silane) trifluoroacetamide (MSTFA) is added, is centrifuged after 40 DEG C of water-baths
Take supernatant to be fitted into GC sample injection bottles, be stored at room temperature；
4. GC-MS is detected：Using GC-MS, to step, 3. gained sample carries out qualitative and quantitative Treatment, obtains Rhodococcus sp
Metabolin is qualitative and quantitative data.
Step (2) includes the extraction to substrate after degraded, GC detections, and specific method is：
1. substrate extracts after degraded：Be inoculated with respectively Rhodococcus sp to step (1) 1. described in degraded in three groups of culture mediums
Experiment；Phase after degradation, extracted 2 times with same volume dichloromethane；After dichloromethane volatilization completely, chromatographic pure dichloromethane is used
After redissolution degradation rate is surveyed with GC；Bacterium culture medium is not added with as control；
2. GC is detected：Using GC, to step, 1. gained sample quantitative determines.Substrate is dropped so as to obtain Rhodococcus sp
The quantitative data of solution.
Step (3) principal component analytical method is：MZmine softwares are imported to the metabolin relative amount data obtained in (1)
Batch processing is carried out, obtains the initial qualitative, quantitative data of metabolin；The primary data of every collection of illustrative plates is, it is necessary to be standardized place
Reason, will each in the peak area of metabolin divided by the collection of illustrative plates internal standard compound phenanthrene peak area, obtain the relatively rich of every kind of metabolin
Spend standardized data.By contrast, select different groups of data to differ larger key metabolites and carry out subsequent analysis, Key Metabolic
Thing includes：(these metabolite content differences can influence microorganism pair to the metabolins such as alanine, methylamine, Heptadecanoic acide and citric acid
The degradation process of pyrene).Finally standardized data is imported in SIMCA13.0 and carries out principal component analysis.
(4) process analysis procedure analysis
Chart is made according to condition of culture difference in the data of step (2) (3), observes and analyzes these metabolin differences,
And then the metabolin and associated metabolic network to be played a crucial role in polycyclic aromatic hydrocarbon pyrene biodegradation process is found, so as to be to carry
Rhodococcus sp condition of culture for the purpose of high polycyclic aromatic hydrocarbon pyrene degradation rate provides optimization direction.
The present invention discloses Rhodococcus sp degrading polycyclic aromatic hydrocarbons pyrene using metabolism group means and combination difference significance analysis
Metabolin difference, further explain the metabolic alterations mechanism related to polycyclic aromatic hydrocarbon pyrene degraded.By to Rhodococcus sp in different lifes
Degraded pyrene ability and metaboilic level under long environment are analyzed, and the metabolin related to degraded pyrene are found, so as to optimize its drop
Solution condition, the raising for polycyclic aromatic hydrocarbon pyrene degradation rate provide direction.This method is alternatively other degrading polycyclic aromatic hydrocarbons microorganism drops
The optimizing research of solution condition provides new idea and method.
Brief description of the drawings
(" A " represents using drop of the pyrene as sole substrate to pyrene degraded situation of the Rhodococcus sp to substrate under Fig. 1 difference degradation conditions
Solution rate；" B " is represented using the degradation rate of pyrene and hexadecane as substrate to pyrene；" C " represents to align by sole substrate of hexadecane
The degradation rate of hexadecane；" D " is represented using the degradation rate of pyrene and hexadecane as substrate to hexadecane).
Fig. 2 difference degradation condition lower eyelid intracellular metabolite thing PCA shot charts；
Fig. 3 difference degradation condition lower eyelid intracellular metabolite thing PCA load diagrams；
The relative abundance of the lower four kinds of biomarkers of Fig. 4 difference degradation conditions.
Embodiment
Below according to the method for the present invention, the present invention will be further described in conjunction with specific embodiments：
Embodiment 1
Rhodococcus sp degradation condition is improved based on metabolism group to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate, including following step
Suddenly：
(1) metabolin of Rhodococcus sp intracellular is measured；
(2) ability of Rhodococcus sp degraded pyrene is analyzed；
(3) principal component analysis
(4) process analysis procedure analysis
Specific method is as follows：
Step (1) includes cell and collects and be quenched, extracts endocellular metabolism thing, metabolin derivatization and GC-MS detections, tool
Gymnastics is made as follows：
1. cell is collected and is quenched：
Rhodococcus sp to culture medium 2, culture medium 3, culture medium 4 is inoculated with respectively and carries out degradation experiment, and different substrate cultivation bases are extremely
3 Duplicate Samples are done less.Phase after degradation, each culture medium sample 20mL, each 3 parts of culture medium, samples taken and 20mL quenched
10min is placed after the liquid that goes out is well mixed, in 5000rpm, 4 DEG C, 5min is centrifuged, abandons supernatant；Removed with 4 DEG C of distillation water washings thin
Medium component on born of the same parents, retain thalline.
The liquid that is quenched is the methanol aqueous solution that volumetric concentration is 60%.
The culture medium 1 is：NH4NO32.1g/L, K2HPO41g/L, kH2PO41g/L, MgSO4·7H2O 0.5g/L,
FeSO4·7H2O 0.01g/L, CaCl20.02g/L, NaCl 5g/L, surplus are water, adjust pH=7.2, sterilizing；Culture medium 2 is：
It is sole carbon source that 100mg/L pyrenes are added in culture medium 1；Culture medium 3 is：2% (v/v) hexadecane is added in culture medium 1
For sole carbon source；Culture medium 4 is：It is carbon source that 100mg/L pyrenes and 2% (v/v) hexadecane are added in culture medium 1.
2. extract endocellular metabolism thing：
The cell for taking step 1. to obtain adds liquid nitrogen grinding in mortar, weighs 30mg and is placed in the good cell of liquid nitrogen grinding
In 1.5mL centrifuge tubes, the extract solution that 0.5mL temperature is -40 DEG C is added, frozen-thawed 3 times after vortex vortex mixer fully mixes
To freeze thawing liquid；By freeze thawing liquid at 4 DEG C, 5000rpm, centrifugation 5min, supernatant is taken to deserve to be called clear liquid a into another centrifuge tube；Lower floor
Cell adds the extract solution that 0.5mL temperature is -40 DEG C, after vortex vortex mixer fully mixes, 4 DEG C, 8000rpm, centrifugation 5min,
Take supernatant to deserve to be called clear liquid b, supernatant a and b are well mixed to obtain supernatant c, 8000rpm, 4 DEG C, 5min is centrifuged, takes 260 μ L
Supernatant c goes to 1.5mL centrifuge tubes, adds the dichloromethane phenanthrene solution that 40uL concentration is 500mg/L, and vortex vortex mixer is fully mixed
After even, under the conditions of -40 DEG C, vacuum freeze drying obtains sample.
The extract solution is that volumetric concentration is 50% methanol aqueous solution.
3. metabolin derivatization
The pyridine that the methoxyl group ammonium salt hydrochlorate that 80 μ L concentration are 20mgmL-1 is added in the sample 2. obtained to step is molten
Liquid, vortex concussion mix sample dissolving, are placed in 40 DEG C of water-bath 80min in water-bath；Add 80 μ L N- methyl-N-
(trimethyl silane) trifluoroacetamide (MSTFA), it is vortexed and mixes, be placed in 40 DEG C of water-bath 80min；Sample 8000rpm, centrifugation
5min, take the μ L of supernatant 100 after centrifugation to be fitted into GC sample injection bottles, be stored at room temperature 2h.
4. GC-MS is detected
Using GC-MS, to step, 3. gained sample carries out qualitative and quantitative Treatment, so as to obtain the metabolin of Rhodococcus sp
Qualitative and quantitative data；
Step (2) includes the extraction to substrate after degraded, GC detections, and specific method is：
1. substrate extracts after degraded
Rhodococcus sp to culture medium 2, culture medium 3, culture medium 4 is inoculated with respectively and carries out degradation experiment, and different substrate cultivation bases are extremely
3 Duplicate Samples are done less.Phase after degradation, each culture medium sample 50mL, then with same volume analysis absolute dichloromethane extraction 2
It is secondary.After dichloromethane volatilization completely, redissolved with chromatographic pure dichloromethane, concentration is in 100-500ppm, excessively 0.22 μm after making redissolution
After organic phase filter membrane, it is fitted into 2mL GC-MS auto injection bottles, degradation rate is surveyed with GC.Bacterium culture medium is not added with as control.
2. GC is detected
Using GC, to step, 1. gained sample quantitative determines.
Step (3) principal component analytical method is：MZmine softwares are imported to the metabolin relative amount data obtained in (1)
Batch processing is carried out, by Mass Spectrometer Method, chromatographic peak structure, a series of steps such as chromatographic peak is deconvoluted, base peak is alignd, peak is identified
The rapid final acquisition initial qualitative, quantitative data of metabolin.The primary data of every collection of illustrative plates, will be every, it is necessary to be standardized
The luxuriant and rich with fragrance peak area of internal standard compound, obtains the relative abundance normalized number of every kind of metabolin in the peak area of individual metabolin divided by the collection of illustrative plates
According to.By contrast, select different groups of data to differ larger key metabolites and carry out subsequent analysis, key metabolites include：Third
(these metabolite content differences can influence degraded of the microorganism to pyrene to the metabolins such as propylhomoserin, methylamine, Heptadecanoic acide and citric acid
Journey).Finally standardized data is imported in SIMCA13.0 and carries out principal component analysis.
(4) process analysis procedure analysis
Rhodococcus sp to quantitative data such as Fig. 1 of degradation of substrates, after degraded 5 days in each system substrate degraded situation,
The degradation rate of pyrene and hexadecane is 55.35% and 96.75% respectively in single substrate system, but both drops in mixed system
Solution rate is respectively then 80.79% and 83.39%, i.e. the degradation rate compared to single substrate system pyrene improves 25.44%.By SPSS
Software is analyzed, and in pyrene with hexadecane difference substrate system, target substrates degradation rate significant difference, that is, changes substrate combination
Influence to degraded is obvious.
Fig. 2 is PCA shot charts, it can be seen that sample is substantially divided into 3 groups, and this shows that different substrate cultivation condition hypothalluses are thin
Really obvious difference be present in the metabolism of born of the same parents' intracellular.Fig. 3 is PCA load diagrams, as can be seen from the figure mainly has four kinds of materials remote
Central point, they are the key metabolites for influenceing 3 packets on shot chart, also referred to as biomarker, and they are followed successively by third
Propylhomoserin, methylamine, Heptadecanoic acide and citric acid, this several material has significant difference in three experimental groups, as Fig. 4 represents this
A little relative abundances of the biomarker in different carbon source system cell metabolite, with abundance sign target substance in the cell
Accumulation.As can be seen from the figure the accumulation difference of these materials is obvious under different carbon sources selection pressure.
Analysis is understood above, the toxicity inhibition cellular respiration of substrate, influences the degraded of pollutant.Reduce substrate poison
Property, improving thalline surrounding environment can promote cell growth to breed, and strengthen absorb ability of the cell to pollutant, preferably
Play biological prosthetic effect of the microorganism to pollution environment.
Embodiment 2
Rhodococcus sp degradation condition is improved based on metabolism group to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate, including following step
Suddenly：
(1) metabolin of Rhodococcus sp intracellular is measured；
(2) ability of Rhodococcus sp degraded pyrene is analyzed；
(3) principal component analysis
(4) process analysis procedure analysis
Specific method is as follows：
Step (1) includes cell and collects and be quenched, extracts endocellular metabolism thing, metabolin derivatization and GC-MS detections, tool
Gymnastics is made as follows：
1. cell is collected and is quenched：
Rhodococcus sp to culture medium 2, culture medium 3, culture medium 4 is inoculated with respectively and carries out degradation experiment, and different substrate cultivation bases are extremely
3 Duplicate Samples are done less.Phase after degradation, each culture medium sample 15-20mL, each culture medium 2-3 parts, by samples taken with
15-20mL is quenched after liquid is well mixed and places 5-10min, in 3000-5000rpm, 4 DEG C, centrifuges 3-5min, abandons supernatant；With 4
DEG C distillation water washing remove cell on medium component, retain thalline.
The liquid that is quenched is the methanol aqueous solution that volumetric concentration is 60%.
The culture medium 1 is：NH4NO32.1g/L, K2HPO41g/L, kH2PO41g/L, MgSO4·7H2O 0.5g/L,
FeSO4·7H2O 0.01g/L, CaCl20.02g/L, NaCl 5g/L, surplus are water, adjust pH=7.2, sterilizing；Culture medium 2 is：
It is sole carbon source that 100mg/L pyrenes are added in culture medium 1；Culture medium 3 is：1% (v/v) value alkane is added in culture medium 1 as only
One carbon source；Culture medium 4 is：It is carbon source that 100mg/L pyrenes and 1% (v/v) value alkane are added in culture medium 1.
2. extract endocellular metabolism thing：
The cell for taking step 1. to obtain adds liquid nitrogen grinding in mortar, weighs 30mg and is placed in the good cell of liquid nitrogen grinding
In 1.5mL centrifuge tubes, the extract solution that 1mL temperature is -40 DEG C is added, frozen-thawed obtains for 4 times after vortex vortex mixer fully mixes
Freeze thawing liquid；By freeze thawing liquid at 4 DEG C, 3000rpm, centrifugation 3min, supernatant is taken to deserve to be called clear liquid a into another centrifuge tube；Lower floor is thin
Born of the same parents add the extract solution that 1mL temperature is -40 DEG C, after vortex vortex mixer fully mixes, 4 DEG C, 10000rpm, centrifugation 3min, take
Supernatant deserves to be called clear liquid b, and supernatant a and b are well mixed to obtain into supernatant c, 10000rpm, 4 DEG C, 3min is centrifuged, takes on 260 μ L
Clear liquid c goes to 1.5mL centrifuge tubes, adds the dichloromethane phenanthrene solution that 40uL concentration is 500mg/L, and vortex vortex mixer fully mixes
Afterwards, under the conditions of -40 DEG C, vacuum freeze drying obtains sample.
The extract solution is that volumetric concentration is 50% methanol aqueous solution.
3. metabolin derivatization
The pyridine that the methoxyl group ammonium salt hydrochlorate that 80 μ L concentration are 20mgmL-1 is added in the sample 2. obtained to step is molten
Liquid, vortex concussion mix sample dissolving, are placed in 40 DEG C of water-bath 80min in water-bath；Add 80 μ L N- methyl-N-
(trimethyl silane) trifluoroacetamide (MSTFA), it is vortexed and mixes, be placed in 40 DEG C of water-bath 80min；Sample 10000rpm, from
Heart 3min, take the μ L of supernatant 100 after centrifugation to be fitted into GC sample injection bottles, be stored at room temperature 2h.
4. GC-MS is detected
With the step of embodiment 1 (1) 4..
Step (2) includes the extraction to substrate after degraded, GC detections, and specific method is：
1. substrate extracts after degraded
Rhodococcus sp to culture medium 2, culture medium 3, culture medium 4 is inoculated with respectively and carries out degradation experiment, and different substrate cultivation bases are extremely
3 Duplicate Samples are done less.Phase after degradation, each culture medium sample 70mL, then with same volume analysis absolute dichloromethane extraction 2
It is secondary.After dichloromethane volatilization completely, redissolved with chromatographic pure dichloromethane, concentration is in 100-500ppm, excessively 0.22 μm after making redissolution
After organic phase filter membrane, it is fitted into 2mL GC-MS auto injection bottles, degradation rate is surveyed with GC.Bacterium culture medium is not added with as control.
2. GC is detected
With the step of embodiment 1 (2) 2..
Step (3) principal component analytical method is：MZmine softwares are imported to the metabolin relative amount data obtained in (1)
Batch processing is carried out, by Mass Spectrometer Method, chromatographic peak structure, a series of steps such as chromatographic peak is deconvoluted, base peak is alignd, peak is identified
The rapid final acquisition initial qualitative, quantitative data of metabolin.The primary data of every collection of illustrative plates, will be every, it is necessary to be standardized
The luxuriant and rich with fragrance peak area of internal standard compound, obtains the relative abundance normalized number of every kind of metabolin in the peak area of individual metabolin divided by the collection of illustrative plates
According to.By contrast, select different groups of data to differ larger key metabolites and carry out subsequent analysis, key metabolites include：Third
(these metabolite content differences can influence degraded of the microorganism to pyrene to the metabolins such as propylhomoserin, methylamine, Heptadecanoic acide and citric acid
Journey).Finally standardized data is imported in SIMCA13.0 and carries out principal component analysis.
(4) process analysis procedure analysis
Chart is made according to condition of culture difference in the data of step (2) (3), observes and analyzes these metabolin differences,
And then the metabolin and associated metabolic network to be played a crucial role in polycyclic aromatic hydrocarbon pyrene biodegradation process is found, so as to be to carry
Rhodococcus sp condition of culture for the purpose of high polycyclic aromatic hydrocarbon pyrene degradation rate provides optimization direction.
It is experimentally confirmed, it is as a result similar to Example 1.
Claims (6)
1. Rhodococcus sp degradation condition is improved to improve the method for polycyclic aromatic hydrocarbon pyrene degradation rate based on metabolism group, it is characterized in that step
It is as follows：
(1) metabolin of Rhodococcus sp intracellular is measured；
(2) ability of Rhodococcus sp degraded pyrene is analyzed；
(3) principal component analysis；
(4) process analysis procedure analysis.
2. the method as described in claim 1, it is characterized in that metabolin be measured side of the step (1) to Rhodococcus sp intracellular
Method is：Degraded using different alkane and pyrene as substrate, the degraded later stage is measured to the metabolin of Rhodococcus sp intracellular, wherein wrapping
Cell is included to collect and be quenched, extract endocellular metabolism thing, metabolin derivatization, GC-MS detections.
3. method as claimed in claim 2, it is characterized in that the methanol aqueous solution that liquid is 60%, the metabolin internal standard of addition is quenched
For dichloromethane phenanthrene solution, derivatization reagent is the pyridine solution and N- methyl-N- (trimethyl silane) of methoxyl group ammonium salt hydrochlorate
Trifluoroacetamide.
4. the method as described in claim 1, it is characterized in that the ability of the pyrene of being degraded to Rhodococcus sp of step (2) carries out analysis method
For：To under different substrates Rhodococcus sp degraded pyrene ability analyze, including substrate after degraded is extracted, GC examine
Survey.
5. method as claimed in claim 4, extract used in its feature is dichloromethane.
6. the method as described in claim 1, it is characterized in that the step (3) principal component analysis is in SIMCA13.0 softwares pair
Metabolin carries out principal component analysis, concretely comprises the following steps and is criticized the metabolin relative amount data importing MZmine softwares of acquisition
Amount processing, by the way that SIMCA13.0 softwares carry out Mass Spectrometer Method, chromatographic peak is built, chromatographic peak deconvolutes, base peak aligns, peak reflects
It is fixed, it is final to obtain the initial qualitative, quantitative data of metabolin.
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