CN109097310A - Degrading polycyclic aromatic hydrocarbons-pyrene anaerobic type bacterial strain and its screening technique and application - Google Patents
Degrading polycyclic aromatic hydrocarbons-pyrene anaerobic type bacterial strain and its screening technique and application Download PDFInfo
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Abstract
The present invention relates to the applications in polycyclic aromatic hydrocarbon in one plant of anaerobism bacterial strain Clostridium sp.LZ25 and its processing industrial pollutants；Fusobacterium, deposit number are accredited as according to its physio-biochemical characteristics and 16S rDNA sequence are as follows: CGMCC NO:15046.Bacterial strain Clostridium sp.LZ25 is anaerobic type microorganism, and culture 30d is to the degradation efficiency of pyrene up to 45% in the inorganic salt liquid culture medium that concentration containing pyrene is 50mg/L；It increases the degradation rate of pyrene with the increase for connecing bacterium amount, reduces with the raising of initial concentration, while larger to the adaptability of pH and temperature.The present invention is based on the metabolic pathways that metabolite deduces Clostridium sp.LZ25 anaerobic degradation pyrene, have important application value to the improvement of the pollutant containing polycyclic arene compound.
Description
Technical field
The invention belongs to the biodegradable processing technology fields of contaminated sludge and sewage, and in particular to a kind of degradation is polycyclic
Aromatic hydrocarbons-pyrene anaerobism bacterial strain and its screening, identification and application.
Background technique
Pyrene is one kind of polycyclic aromatic hydrocarbon (PAHs), due to high carcinogenic, mutagenicity and teratogenesis, for a long time always by
It is classified as Priority-Control Organic Pollutants listed by Environmental Protection Agency USA.Polycyclic aromatic hydrocarbon has as persistence most common in environment
One of machine pollutant is mainly formed in combustion of fossil fuel, biomass combustion and crude oil release, petroleum and coai-tar product.It releases
The polycyclic aromatic hydrocarbon being put into environment can be removed mainly by many methods, including volatilization, photooxidation, chemical oxidation, biology are tired
Product, biodegrade and absorption.Wherein, biodegrade is to remove the most thorough of polycyclic aromatic hydrocarbon and endanger the smallest method, it has also become
The main direction of studying of pollution control.
During seeking degrading polycyclic aromatic hydrocarbons, it is polycyclic aromatic hydrocarbon microbial degradation that efficient degrading bacterial strain is isolated in screening
The key of technology.Relative to the PAHs of low molecular weight, high molecular weight (HMW) PAHs more than Fourth Ring and Fourth Ring is due to its molecule knot
Structure is complicated, be difficult to be oxidized, poorly water-soluble etc. is difficult to filter out the bacterial strain of efficient degradation.Pyrene is a kind of four with symmetrical structure
Cycloaromatics is the research polycyclic virtue of high molecular weight because its is rich and its structural similarity with other polycyclic aromatic hydrocarbon with high molecular weight
The biodegradable good model compound of hydrocarbon.Have now been found that the typical bacteria for the PAHs that can degrade under aerobic conditions has very much
Kind, and the aerobic degradation mechanism of 2-5 ring polycyclic aromatic hydrocarbon is studied.Although degradation pathway of the PAHs under aerobic conditions
It is proposed extensively, but the research for exploring anaerobic degradation approach is limited, particular for high molecular weight PAHs.Therefore, to Fourth Ring and
The research of PAHs more than Fourth Ring also has been a hot spot of research at present, especially the degradation to PAHs more than Fourth Ring and Fourth Ring
Approach still needs to deeply probe into.
Summary of the invention
Goal of the invention:
1, at high cost, no for being removed in industrial sludge and sewage containing higher concentration toxic organic pollutant polycyclic aromatic hydrocarbon
Thorough problem provides the anaerobism bacterial strain and screening technique of a kind of degrading polycyclic aromatic hydrocarbons-pyrene, and raw in morphological feature, physiology
Change and pyrene degradation bacteria strains are identified on molecular level.
2, a kind of application of anaerobism bacterial strain during pyrene degradation treatment is provided.
Technical solution:
The anaerobe of the efficient degradation pyrene is clostridium, classification naming Clostridium
Sp.LZ25 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC), and preservation is compiled
Number are as follows: CGMCC NO:15046, preservation date are as follows: on December 11st, 2017, preservation address are as follows: the Chinese Beijing Chinese Academy of Sciences
Institute of microbiology.
The screening technique of Clostridium sp.LZ25 of the present invention: it is trained by the inorganic salts of sole carbon source of pyrene
It supports in base, separation screening is carried out to pyrene degradation bacteria strains from oil-containing petrochemical sludge.
It specifically, is screening target with the flora in oil-containing petrochemical sludge, using the minimal medium containing pyrene as Jie
Matter, continuous acclimating have the anaerobism bacterial strain of pyrene Utilization ability, enriched medium dilution spread to LB solid medium are detested
Oxygen culture, the different pyrene degradation bacteria strains of picking form carry out scribing line separation obtain pure culture, select wherein to pyrene degradation efficiency most
High bacterial strain, as Clostridium sp.LZ25.
More specifically steps are as follows；
(1) using the flora of petrochemical sludge containing polycyclic aromatic hydrocarbon as screening target, fresh sludge is taken to vibrate in physiological saline；
(2) the pyrene acetone soln that organic filter membrane is already expired is added into sterilized anaerobism bottle, opening stands overnight removing third
Ketone；
(3) sterilized minimal medium is added in the anaerobism bottle in step (2) in a nitrogen atmosphere；
(4) it by the suspension in step (1), is accessed in step (3) using 10% inoculum concentration using pyrene as the inorganic of sole carbon source
In salt culture medium, constant-temperature shaking culture；Then it is transferred to again and continues to cultivate in the minimal medium containing pyrene；So repeat,
Until pyrene concentration is 200mg/L in culture medium；
(5) culture solution of last time is diluted and is applied to Anaerobic culturel on LB plating medium, plate draws lines repeatedly
Separation, until obtaining purebred bacterium Clostridium sp.LZ25.
Specifically, the minimal medium is by NaCl 24g/L, KCl 0.7g/L, MgSO4·7H20 0.7g/L、
NH4Cl 1g/L、NaNO3 0.5g/L、KH2PO4 2g/L、Na2HPO43g/L adds water to 1.0L preparation, pH value 6.5-7.5.LB
Culture medium: NaCl 10g/L, peptone 10g/L, yeast powder 5g/L.PH value is adjusted to 7.0, solid medium be added agar 15~
20g/L, 121 DEG C of sterilizing 20min.
The identification method of Clostridium sp.LZ25 of the present invention:
Clostridium sp.LZ25 is very fast in LB cultured on solid medium, and 30 DEG C, it is 1mm's that 72h, which can form diameter,
Yellow color colonies, colony edge is neat, matt, prominent, sticky, moistens, smooth, opaque, thallus in elongated rod shape (1.02 ×
2.0μm).Biolog Automatic Analyzer for Microbes tests 94 kinds of biochemical phenotypes of Clostridium sp.LZ25, and
Identify strain.71 utilization of carbon source measurements and 23 chemosensitivity measurements are analyzed in 94 phenotype tests.By micro point
Analysis system GENIII MicroPlateBiolog determines that important biochemical characteristics is as shown in table 1.Wherein Clostridium
Sp.LZ25 can be efficiently used in 9 kinds of carbon sources, including dextrin, raffinose, D- sorbic acid, D- galacturonic acid, L- galacturonic acid
Ester, D- glucuronic acid, glucuronamide, α-batanone acid and acetoacetate.In chemosensitivity test simultaneously, to 5 kinds of chemicals
Matter is sensitive, can give birth to well including 1% sodium lactate and acidum nalidixicum, and at pH6,1%NaCl and 4%NaCl
It is long.The result of bacterial strain LZ25 Physiology and biochemistry identification and the feature of Clostridium are closest.
Specifically, the pure culture of acquisition is seeded on the BUG culture medium that Biolog is recommended, in IF-A inoculation liquid
Bacteria suspension is prepared, microwell plate data are read on Biolog readout instrument, reading is repeated 3 times, finally compares identification with database again.
Identification method on the pyrene degradation bacteria strains Clostridium sp.LZ25 molecular level: expanded using universal primer
Increase the 16S rDNA partial sequence of bacterial strain LZ25.Segment obtained is sequenced, ncbi database compares the 16S rDNA
Sequence on a molecular scale identifies bacterial strain LZ25 to Clostridium.
The 16S rDNA PCR amplification condition is following (enzyme used is ExTaq): 95 DEG C of initial denaturation 5min, 95 DEG C of denaturation
30s, 55 DEG C of annealing 30s, 72 DEG C of extension 90s, 30 circulations, 72 DEG C of extension 5min, 1.0% agarose gel electrophoresis, which confirms, to be expanded
The 16S rDNA segment of increasing.
The invention further relates to the anaerobism bacterial strain Clostridium sp.strain LZ25 (to be also referred to simply as below
LZ25) application during pyrene degradation treatment, the specific steps are as follows:
(1) bacterial strain Clostridium sp.Z25 seed liquor culture: bacterial strain Clostridium sp.LZ25 seed liquor uses
LB liquid medium, the bacterial strain Clostridium sp.LZ25 for plate preservation of making even choose bacterium colony with oese in anaerobic operation platform
Shaken cultivation in the anaerobism bottle containing LB liquid medium is accessed, seed liquid concentrate is collected after centrifugation as degradation kind in thallus
Sub- liquid；
(2) bacterial strain Clostridium sp.LZ25 degradation pyrene: being added minimal medium in the pollutant containing pyrene,
The seed liquor that step (1) obtains is accessed, carries out degradation reaction under anaerobic condition.
Degradation reaction is carried out under anaerobic condition in the step (2), access seed liquid measure is 1%-20%, preferably
5-20%.
Degradation reaction is carried out under anaerobic condition in the step (2), the pH value of reaction is 4.8-9.6, preferred pH
Value is 6.0-8.4.
Carry out degradation reaction under anaerobic condition in the step (2), range of reaction temperature between 22 DEG C -50 DEG C,
Preferably 30-40 DEG C.
Degradation reaction is carried out under anaerobic condition in the step (2), reaction substrate pyrene concentration range is in 50mg/L-
Between 200mg/L, preferably 50-100mg/L.
According to pyrene degradation bacteria strains Clostridium sp.LZ25 degrade during the cultivation process intermediate supersession caused by pyrene produce
Object, thus it is speculated that go out the metabolic pathway of strains for degrading pyrene, the specific steps are as follows:
(1) pyrene degradation bacteria strains Clostridium sp.LZ25 cultivates 40d in optimal conditions, passes through liquid phase color every 10d
Pyrene residual quantity in spectrum measurement culture.
(2) the relatively high system of pyrene degradation rate is chosen, the intermediate supersession in Gc-mss pyrene degradation process is passed through
Product.
(3) it in mesostate, selects the representative are machine objects as substrate, detects degradation of the bacterial strain to it
Rate, and analyze metabolite.
(4) comprehensive analysis mesostate, according to the metabolic pathway of analysis of the molecular structure strains for degrading pyrene.
Beneficial effects of the present invention: anaerobic bacteria Clostridium sp.LZ25 of the invention can be right under anaerobic
Polycyclic aromatic hydrocarbon, especially pyrene are degraded, and degradation efficiency with higher, in the processing applied to industrial sludge and sewage,
A kind of practicable side is provided to solve the problems, such as that the removal of higher concentration organic pollutant polycyclic aromatic hydrocarbon is at high cost, halfway
Method.It specifically includes:
1, the bacterial strain is high-efficient to the anaerobic degradation of pyrene, and with the increase of inoculum concentration, the degradation rate of pyrene is obviously increased.
2, bacterial strain LZ25 is wider to the adaptation range of temperature when degrading pyrene, adapts to the dense of more wide pH range and pyrene
Spend range.
3, the present invention is for having important application value in the improvement of the pollutant containing polycyclic arene compound.
Detailed description of the invention
Clostridium sp.LZ25 phylogenetic tree of the Fig. 1 based on 16S rDNA gene homology；
The pyrene degradation curve and its growth curve of Fig. 2 bacterial strain LZ25；
The pyrene degradation characteristic of Fig. 3 a bacterial strain LZ25, inoculation compare the influence of bacterial strain LZ25 degradation pyrene；
Influence of Fig. 3 b temperature to bacterial strain LZ25 degradation pyrene；
Influence of Fig. 3 c pH to bacterial strain LZ25 degradation pyrene；
Influence of Fig. 3 d initial substrate concentration to bacterial strain LZ25 degradation pyrene；
Fig. 4 Clostridium sp.LZ25 analyzes the GC-MS of pyrene intermediates；
Fig. 5 Clostridium sp.LZ25 is to luxuriant and rich with fragrance degradation curve；
Fig. 6 Clostridium sp.LZ25 analyzes the GC-MS of the intermediate product of phenanthrene degradation；
Approach of Fig. 7 Clostridium sp.LZ25 to pyrene anaerobic degradation.
Specific embodiment
Detailed description of the preferred embodiments below, it should be noted however that protection of the invention
Range is not limited to these specific embodiments, but is determined by claims.
Experimental method used in following embodiments is conventional method without specified otherwise, used experiment reagent
Consumptive material etc. can be bought without specified otherwise from commercial use.
Embodiment one
The separation screening of pyrene degradation bacteria strains Clostridium sp.LZ25:
(1) using the flora of petrochemical sludge containing polycyclic aromatic hydrocarbon as screening target, fresh sludge 10g and 100mL 0.85% are taken
Physiological saline in vibrate 20min.
(2) the pyrene acetone soln that 0.22 μm of organic filter membrane is already expired is added into the anaerobism bottle for sterilizing 20min through 121 DEG C, is
Acetone is removed to stand overnight anaerobism bottle opening.
(3) volume is to add in the anaerobism bottle of 100mL or 200mL in step (2) in the anaerobic operation platform full of nitrogen
Enter the sterilized minimal medium of 50mL or 100mL (pH=6.5-7.5), and 0.1% resazurin solution is added and refers to as oxygen
Show agent and cysteine as oxygen scavenger, finally pours nitrogen sealing.Wherein minimal medium: NaCl 24g/L,
KCl0.7g/L、MgSO4·7H20 0.7g/L、NH4Cl 1g/L、NaNO3 0.5g/L、KH2PO4 2g/L、Na2HPO43g/L；Its
Middle pyrene concentration is 20mg/L-200mg/L.LB culture medium: NaCl 10g/L, peptone 10g/L, yeast powder 5g/L.LB culture medium
LB plating medium is made in 15~20g/L of middle addition agar.The above culture medium uses high-pressure steam sterilizing pan in 121 DEG C of conditions
Lower sterilizing 20min.
(4) by the suspension in step (1), being accessed in step (3) with 10% inoculum concentration is unique with pyrene (10mg/L)
In the minimal medium of carbon source, 30 DEG C of oscillation rates of constant temperature are that 180r/min cultivates 7d.Then it is transferred to 20mg/L's again
In PAHs minimal medium, continue to cultivate.It so repeats, until PAHs concentration is 200mg/L in culture medium.
(5) culture solution of last time is carried out 10-1To 10-9Gradient dilution is simultaneously applied on LB plating medium, is passed through
Anaerobic culturel bag culture, 30 DEG C of Anaerobic culturel 3d pick out growth single colonie rapid, abundant, and progress plate draws lines repeatedly to be divided
From until obtaining purebred bacterium.To prevent pyrene by photodissociation, above-mentioned steps are operated in the incubator of darkroom.
(6) scribing line separation obtains pure culture bacterial strain, selects wherein to the highest bacterial strain of pyrene degradation efficiency, is named as bacterial strain
LZ25。
(7) bacterial strain LZ25 is inoculated in using 50mg/L pyrene as in the liquid inorganic salt culture medium of sole carbon source, in 30 DEG C,
After 180r/min shaking table culture 14d, destructiveness sampling, after being extracted with dichloromethane, after rotary evaporation, with methanol constant volume, mistake
0.22 μm of organic filter membrane, sample are analyzed for high performance liquid chromatography detection.The retention time of pyrene in the control culture solution of bacterium is not connect
For 7min or so, and inoculating strain LZ25 and after cultivating 14d, the content of pyrene is decreased obviously in culture solution.
Embodiment two
The identification of pyrene degradation bacteria strains:
Bacterial strain is very fast in LB cultured on solid medium, and 30 DEG C, 72h can form the yellow color colonies that diameter is 1mm, bacterium colony side
Edge is neat, matt, prominent, sticky, moistens, smooth, opaque, and thallus is in elongated rod shape (1.02 × 2.0 μm).Based on its physiology
Biochemical character (table 1) and 16S rRNA sequence, bacterial strain LZ25 are accredited as Clostridium category.Finally that homology is high bacterium
After the base sequence of strain is successively downloaded, similarity analysis is carried out with clustaLZ, and use Neighbor- with 4 software of MEGA
Joining method phylogenetic tree construction figure (Fig. 2).
Clostridium sp.LZ25 can efficiently use 9 kinds of carbon sources, including dextrin, raffinose, D- sorbic acid, D- gala
Uronic acid, L- galacturonic acid lactone, D- glucuronic acid, glucuronamide, α-batanone acid and acetoacetate.Chemical-sensitive simultaneously
Property test in, it is sensitive to 5 kinds of chemical substances, including 1% sodium lactate and acidum nalidixicum, and in pH6,1%NaCl and 4%
It can be grown well under NaCl.The result of bacterial strain LZ25 Physiology and biochemistry identification and the feature of Clostridium connect the most
Closely.
The physiological and biochemical property of 1 bacterial strain LZ25 of table measures
Embodiment three
The degradation characteristic of pyrene degradation bacteria Clostridium sp.LZ25, the specific steps are as follows:
(1) bacterial strain LZ25 seed liquor culture: bacterial strain LZ25 seed liquor uses LB liquid medium, the bacterial strain for plate preservation of making even
LZ25 chooses bacterium colony with oese in anaerobic operation platform and accesses the anaerobism bottle containing 50mL LB liquid medium, 30 DEG C of oscillation trainings
Support 72 hours, shaking speed 180r/min, the bacterium solution of acquisition with 10% inoculation than repetition of activation twice after, thallus passes through with 12
000r/min is centrifuged 10 minutes collection seed liquid concentrates, and is resuspended in minimal medium, and OD is made600nm=1.5
Bacteria suspension is as degradation seed liquor；
(2) dynamics of bacterial strain LZ25 degradation pyrene: in the minimal medium that pyrene initial concentration is 50mg/L, by 10%
Inoculum concentration accesses seed liquor, and in 30 DEG C, 180r/min shake culture, destructive sampling, measures OD at regular intervals600And pyrene
Residual concentration.
(3) influence of inoculum concentration and pH to bacterial strain LZ25 degradation pyrene: in the inorganic salts culture that pyrene initial concentration is 50mg/L
In base, respectively by 1%, 5%, 10%, 20% inoculum concentration access seed liquor in 30 DEG C, 180r/min shake culture；Respectively at
Initial pH value 4.2,6.0,7.2,8.4,9.6,30 DEG C, 180r/min shake culture measure inoculum concentration and pH value to bacterial strain LZ25
The influence of degradation pyrene.
(4) influence of cultivation temperature and initial substrate concentration to bacterial strain LZ25 degradation pyrene: being 50mg/L in pyrene initial concentration
Minimal medium in, access seed liquor by 10% inoculum concentration, respectively at 22 DEG C, 30 DEG C, 38 DEG C, 50 DEG C, pH 7.2,
180r/min shake culture；Respectively in the inorganic salts culture that pyrene initial concentration is 50mg/L, 100mg/L, 150mg/L, 200mg/L
In base, seed liquor is accessed by 10% inoculum concentration, in 30 DEG C, 180r/min shake culture, measures cultivation temperature and substrate is initially dense
Spend the influence to bacterial strain LZ25 degradation pyrene.
As seen from Figure 2, bacterial strain LZ25 is inoculated into 5d in culture medium, degradation pyrene is begun to, without apparent retardation
Phenomenon；Degradation rate gradually increases later, to 30d after the pyrene containing 50mg/L degrade to 28mg/L or so.In addition, may be used also from figure
To find out, with the degradation of pyrene, after the increment of bacterial strain LZ25 experienced the stagnation of 5d, start to gradually increase；When pyrene degradation rate
When reaching maximum, the increment of bacterial strain LZ25 also reaches maximum；And in 30d-40d, under the increment of bacterial strain LZ25 starts
Drop.It should be the result shows that bacterial strain LZ25 can use pyrene is grown.
With the increase of inoculum concentration, the degradation rate of pyrene is obviously increased.When inoculum concentration is 1%, degradation rate of the pyrene in 30d
It is 21.2%；And when connecing bacterium amount and reaching 10%, pyrene degradation rate ramp-up rate slows down, and when to connect bacterium amount be 10%, pyrene degradation rate is
45.3%, when connecing bacterium amount and being increased to 20%, degradation rate reaches 50.0% (Fig. 3 a).Clostridium sp.LZ25 is 7.2 in pH
It is essentially identical to the degradation rate of pyrene when with 8.4；And at 6.4, the degradation rate of pyrene is just by opposite inhibition；When pH is 4.8 Hes
When 9.6, the degradation rate of pyrene only only has 10% (Fig. 3 b).Bacterial strain LZ25 is wider to the adaptation range of temperature when degrading pyrene,
Good effect is all had between 30 DEG C and 38 DEG C；Even if the degradation rate of pyrene also can achieve when cultivation temperature is 22 DEG C
29%.This result illustrates that the degrading enzyme that bacterial strain LZ25 is generated has stronger thermal adaptability (Fig. 3 c).Bacterial strain LZ25 is to pyrene
Degradation rate is reduced with the increase of concentration of substrate.When pyrene initial concentration is 50mg/L, it is in the degradation rate of 30d
45.3%；And when pyrene initial concentration is 200mg/L, it still can achieve 14.2% in the degradation rate of 30d, illustrate the bacterial strain
There is efficient degradation efficiency (Fig. 3 d) to pyrene.
Example IV
Degradation pathway of the pyrene degradation bacteria Clostridium sp.LZ25 to pyrene:
Qualitative analysis is carried out by GC-MS, furthers investigate Clostridium sp.LZ25 in pyrene anaerobic degradation process
A series of mesostates.The chromatogram for extracting sample shows Clostridium sp.LZ25 shape in pyrene degradation process
At a variety of metabolites.
As can be known from Fig. 4, it compared with blank control, is found in the reaction system of Clostridium sp.LZ25 in 10d
7 kinds of substances.13.426, the absorption peak of 13.169,11.411 and 13.350min through mass spectrum compare be respectively pyrene (C16H10,
CAS:129-00-0), 4,5- dihydropyrene (C16H12, CAS:6628-98-4), luxuriant and rich with fragrance (C14H10, CAS:85-01-8) and 1,1'-
(1- butenylidene) double benzene (C16H16, CAS:1726-14-3)；7.409, the absorption peak of 10.779 and 23.203min is through mass spectrum
Comparison is paracresol (C7H8O, CAS:106-44-5), original pair tea phenol (C7H6O4, CAS:99-50-3) and 2,4-dis 3-ethyl hexane respectively
Product after (C20H26O2, CAS:85-95-0) silanization；2,4-dis 3-ethyl hexane, paracresol and original pair tea phenol are speculated as 1,1'-, and (1- is sub-
Cyclobutenyl) double benzene the product being sequentially generated after being repeatedly metabolized.It is inoculated with the reaction system of Clostridium sp.LZ25
Have found other two kinds of new metabolites in the metabolite of 20d, the chromatography residence time be respectively 6.575min and
7.993min is accredited as phenol (C6H6O, CAS:108-95-2) and benzyl carbinol (C8H10O, CAS:60-12- after mass spectrum compares
8) product after silanization.
Due to having found phenanthrene in the mesostate of pyrene, we further study degradation using phenanthrene in the medium
Approach.Biodegrade experiment shows that in addition to pyrene, Clostridium sp.LZ25 can also degrade phenanthrene.Clostridium
After sp.LZ25 is cultivated 40 days, 44.4% (Fig. 5) of the degradable 50mg/L phenanthrene of LZ25.Luxuriant and rich with fragrance degradation time is longer.By 30 days
The phenanthrene that initial concentration is 50mg/L is degraded to only 24.5mg/L by culture, Clostridium sp.LZ25.It all confirms above
Clostridium sp.LZ25 can use pyrene and phenanthrene as growth substrate.
In order to further study the degradation pathway of pyrene, qualitative analysis is carried out to luxuriant and rich with fragrance Metabolic Intermediate using GC-MS technology.
Compared with luxuriant and rich with fragrance blank control, GC-MS has analyzed and identified the anaerobic metabolism (Fig. 6) luxuriant and rich with fragrance at the 20th day, in addition to retention time is
The phenanthrene of 11.405min, is also 6.682 in retention time and 7.402min is the derivative after detecting phenol and paracresol silanization
Object.Finally, being detected in the residence time of 15.365min through silanization in luxuriant and rich with fragrance culture in culture in the 30th day (Fig. 6)
2'- hydroxyl -4'- methyl acetophenone (C9H10O2, CAS:6921-64-8) afterwards.The above substance passes through standard items GC- respectively
MS analysis confirms.
According to the analysis of the above intermediate product, approach such as Fig. 7 of Clostridium sp.LZ25 anaerobic degradation pyrene is derived
It is shown.There it can be seen that pyrene is reduced into 4,5- dihydropyrene by the way that two hydrogen atoms are added, the then c-c on saturated carbon atom
Key cutting generates luxuriant and rich with fragrance.Hydro-reduction is considered as assuming that the initial activation reaction of pyrene in approach.Then, phenanthrene is degraded to 1,
1'- (1- butenylidene) double benzene.1,1'- (1- butenylidene) double benzene may be converted into three kinds of products.A kind of approach is 1,1'-
(1- butenylidene) double benzene are converted into 2,4-dis 3-ethyl hexane by hydrolyzing hydroxylating.2,4-dis 3-ethyl hexane is further converted to paracresol, and paracresol is again
Hydroxylating and formed to hydroxy-benzyl alcohol, using to hydroxyl benzyl aldehyde to form phenol.Paracresol be also possible to through hydroxylating and
The carboxylated of methyl is converted into protocatechuic acid.1,1'- (1- butenylidene) double benzene can also form 2 '-by hydrolysis and hydroxylating
Hydroxypropiophenonepreparation, then methylate and to form 1- (2- hydroxy-5-methyl base phenyl) -1- acetone, then carboxylated conversion generates protocatechuic acid.
According to molecular structure, 1,1'- (1- butenylidene) double benzene are also possible to generate a kind of catabolite cinnamyl alcohol, and cinnamyl alcohol further drops
Solution is benzyl carbinol, and benzyl carbinol is eventually converted into phenol.
Claims (10)
1. one plant of anaerobism bacterial strain, which is characterized in that classification naming is Clostridium sp.LZ25, deposit number are as follows: CGMCC
NO:15046。
2. the screening technique of Clostridium sp.LZ25 described in claim 1, which is characterized in that utilize the nothing containing pyrene
Machine salt culture medium has pyrene Utilization ability as medium, from acclimating continuous in petrochemical sludge containing polycyclic aromatic hydrocarbon
Clostridium sp.LZ25 bacterial strain, the specific method is as follows:
(1) using the flora of petrochemical sludge containing polycyclic aromatic hydrocarbon as screening target, fresh sludge is taken to vibrate in physiological saline；
(2) the pyrene acetone soln that organic filter membrane is already expired is added into sterilized anaerobism bottle, opening stands overnight removing acetone；
(3) sterilized minimal medium is added in the anaerobism bottle in step (2) in a nitrogen atmosphere；
(4) by the suspension in step (1), using in 10% inoculum concentration access step (3) using pyrene as the inorganic salts of sole carbon source
In culture medium, constant-temperature shaking culture；Then it is transferred in the minimal medium containing pyrene again, continues to cultivate；It so repeats, directly
Into culture medium, pyrene concentration is 200mg/L；
(5) culture solution of last time is diluted and is applied on LB plating medium, by Anaerobic culturel, plate draws lines repeatedly
Separation, until obtaining purebred bacterium Clostridium sp.LZ25.
3. the screening technique of Clostridium sp.LZ25 according to claim 2, which is characterized in that described is inorganic
Salt culture medium composition is NaCl 24g/L, KCl 0.7g/L, MgSO4·7H20 0.7g/L、NH4Cl 1g/L、NaNO3 0.5g/
L、KH2PO4 2g/L、Na2HPO43g/L, pH value 6.5-7.5.
4. the identification method of Clostridium sp.LZ25 described in claim 1, which is characterized in that according to the micro- life of Biolog
Object automatic analysis system carries out test Preliminary Identification to 94 kinds of biochemical phenotypes of Clostridium sp.LZ25, is drawn using general
Object expands the 16S rDNA partial sequence of bacterial strain Clostridium sp.LZ25, segment obtained is sequenced, NCBI number
The 16S rDNA sequence is compared according to library, on a molecular scale identifies bacterial strain Clostridium sp.LZ25 to category.
5. application of the anaerobism bacterial strain Clostridium sp.LZ25 described in claim 1 in pyrene biodegradation process.
6. application of the anaerobism bacterial strain Clostridium sp.LZ25 according to claim 5 in pyrene biodegradation process,
It is characterized in that, specific step is as follows:
(1) bacterial strain Clostridium sp.LZ25 seed liquor culture: bacterial strain Clostridium sp.LZ25 seed liquor uses LB
Fluid nutrient medium, the bacterial strain Clostridium sp.LZ25 for plate preservation of making even, chooses bacterium colony with oese in anaerobic operation platform and connects
Enter shaken cultivation in the anaerobism bottle containing LB liquid medium, seed liquid concentrate is collected after centrifugation as degradation seed in thallus
Liquid；
(2) bacterial strain Clostridium sp.LZ25 degradation pyrene: minimal medium is added in the pollutant containing pyrene, accesses
The seed liquor that step (1) obtains carries out degradation reaction under anaerobic condition.
7. application of the anaerobism bacterial strain Clostridium sp.LZ25 according to claim 6 in pyrene biodegradation process,
It is characterized in that, access seed liquid measure is 1%-20% in step (2).
8. application of the anaerobism bacterial strain Clostridium sp.LZ25 according to claim 6 in pyrene biodegradation process,
It is characterized in that, the pH value reacted in step (2) is 4.8-9.6.
9. application of the anaerobism bacterial strain Clostridium sp.LZ25 according to claim 6 in pyrene biodegradation process,
It is characterized in that, range of reaction temperature is between 22 DEG C -50 DEG C in step (2).
10. anaerobism bacterial strain Clostridium sp.LZ25 answering in pyrene biodegradation process according to claim 6
With, which is characterized in that reaction substrate pyrene concentration range is between 50mg/L-200mg/L in step (2).
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