CN109097310B - Anaerobic strain for degrading polycyclic aromatic hydrocarbon-pyrene and screening method and application thereof - Google Patents

Anaerobic strain for degrading polycyclic aromatic hydrocarbon-pyrene and screening method and application thereof Download PDF

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CN109097310B
CN109097310B CN201811047432.2A CN201811047432A CN109097310B CN 109097310 B CN109097310 B CN 109097310B CN 201811047432 A CN201811047432 A CN 201811047432A CN 109097310 B CN109097310 B CN 109097310B
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周俊
李想
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Abstract

The invention relates to an anaerobic strain Clostridium sp.LZ25 and application thereof in treating polycyclic aromatic hydrocarbon in industrial pollutants; the strain is identified as the clostridium according to the physiological and biochemical characteristics and the 16S rDNA sequence, and the preservation number is as follows: CGMCC NO: 15046. The strain Clostridium sp.LZ25 is an anaerobic microorganism, and the degradation efficiency of pyrene reaches 45 percent after the strain is cultured for 30 days in an inorganic salt liquid culture medium with pyrene concentration of 50 mg/L; the degradation rate of pyrene is increased along with the increase of inoculation amount, is reduced along with the increase of initial concentration, and has larger adaptability to pH and temperature. The metabolic pathway for anaerobic pyrene degradation of Clostridium sp.LZ25 is deduced based on the metabolic products, and the method has important application value in treatment of pollutants containing polycyclic aromatic hydrocarbon compounds.

Description

Anaerobic strain for degrading polycyclic aromatic hydrocarbon-pyrene and screening method and application thereof
Technical Field
The invention belongs to the technical field of biodegradation treatment of polluted sludge and sewage, and particularly relates to an anaerobic strain for degrading polycyclic aromatic hydrocarbon-pyrene, and screening, identification and application thereof.
Background
Pyrene, a species of Polycyclic Aromatic Hydrocarbons (PAHs), has long been listed as a priority organic pollutant by the united states environmental protection agency due to its high carcinogenicity, mutagenicity, and teratogenicity. Polycyclic aromatic hydrocarbons are one of the most common persistent organic pollutants in the environment, formed primarily from fossil fuel combustion, biomass combustion and crude oil release, petroleum and coal tar products. Polycyclic aromatic hydrocarbons released into the environment can be removed primarily by a number of methods, including volatilization, photooxidation, chemical oxidation, bioaccumulation, biodegradation, and adsorption. Among them, biodegradation is the most thorough and least harmful method for removing polycyclic aromatic hydrocarbons, and has become a main research direction for pollution control.
In the process of exploring degradation of the polycyclic aromatic hydrocarbon, screening and separating out high-efficiency degradation strains is the key of a polycyclic aromatic hydrocarbon microbial degradation technology. Compared with low molecular weight PAHs, High Molecular Weight (HMW) PAHs with four or more rings are difficult to screen out strains with high-efficiency degradation due to the complex molecular structure, difficult oxidation, poor water solubility and the like. Pyrene is tetracyclic aromatic hydrocarbon with a symmetrical structure, and is a good model compound for researching biodegradation of high-molecular-weight polycyclic aromatic hydrocarbon due to the richness and structural similarity of pyrene with other high-molecular-weight polycyclic aromatic hydrocarbon. A variety of typical bacteria capable of degrading PAHs under aerobic conditions are found, and the aerobic degradation mechanism of 2-5-ring polycyclic aromatic hydrocarbons is studied. Although degradation pathways for PAHs under aerobic conditions are widely proposed, research exploring anaerobic degradation pathways has been limited, particularly for high molecular weight PAHs. Therefore, the research on the PAHs with four rings or more is still a hot point of research at present, and particularly, the degradation route of the PAHs with four rings or more needs to be deeply researched.
Disclosure of Invention
The purpose of the invention is as follows:
1. aiming at the problems of high cost and incomplete removal of polycyclic aromatic hydrocarbon containing high-concentration toxic organic pollutants in industrial sludge and sewage, an anaerobic strain for degrading polycyclic aromatic hydrocarbon-pyrene and a screening method are provided, and pyrene degrading strains are identified on morphological characteristics, physiological biochemistry and molecular level.
2. Provides an application of the anaerobic strain in the pyrene degradation treatment process.
The technical scheme is as follows:
the anaerobic microorganism for efficiently degrading pyrene is clostridium, is classified and named clostridium lz25, is preserved in China general microbiological culture Collection center (CGMCC for short), and has the preservation number: CGMCC NO:15046, preservation date is: 12 and 11 in 2017, the preservation address is as follows: institute of microbiology, national academy of sciences, Beijing, China.
The screening method of Clostridium sp.lz25 of the invention comprises the following steps: in an inorganic salt culture medium which takes pyrene as a unique carbon source, pyrene degradation strains are separated and screened from oil-containing petrochemical sludge.
Specifically, the flora in the sludge containing oil and petrochemical is taken as a screening target, an inorganic salt culture medium containing pyrene is taken as a medium, anaerobic strains with pyrene utilization capacity are continuously enriched and domesticated, the enriched culture medium is diluted and coated on an LB solid culture medium for anaerobic culture, pyrene degradation strains with different forms are selected for streak separation to obtain pure culture, and the strain with the highest pyrene degradation efficiency is selected, namely Clostridium sp.LZ25.
The more specific steps are as follows;
(1) taking a petrochemical sludge flora containing polycyclic aromatic hydrocarbon as a screening target, and taking fresh sludge to oscillate in normal saline;
(2) adding pyrene acetone solution which is filtered by an organic filter membrane into a sterilized anaerobic bottle, and opening the bottle to stand overnight to remove acetone;
(3) adding a sterilized inorganic salt culture medium into the anaerobic bottle in the step (2) under a nitrogen atmosphere;
(4) inoculating the suspension in the step (1) into an inorganic salt culture medium which takes pyrene as a unique carbon source in the step (3) by using an inoculation amount of 10%, and carrying out constant-temperature shaking culture; then inoculating the culture medium to an inorganic salt culture medium containing pyrene again for continuous culture; repeating the steps until the concentration of pyrene in the culture medium is 200 mg/L;
(5) diluting the last culture solution, coating the diluted culture solution on an LB plate culture medium for anaerobic culture, repeatedly drawing lines on the plate, and separating until pure strain Clostridium sp.LZ25 is obtained.
Specifically, the inorganic salt culture medium is prepared from NaCl 24g/L, KCl 0.7.7 g/L, MgSO4·7H 20 0.7g/L、NH4Cl 1g/L、NaNO30.5g/L、KH2PO42g/L、Na2HPO43g/L, adding water to 1.0L for preparation, and the pH value is 6.5-7.5. LB culture medium: NaCl 10g/L, peptone 10g/L and yeast powder 5 g/L. Adjusting the pH value to 7.0, adding agar 15-20 g/L into the solid culture medium, and sterilizing at 121 ℃ for 20 min.
The identification method of Clostridium sp.lz25 comprises the following steps:
clostridium sp.lz25 grows faster on LB solid medium, forms yellow colonies with a diameter of 1mm at 30 ℃, 72h, has clean edges, is matt, outstanding, sticky, moist, smooth, opaque, and has a long rod shape (1.02 × 2.0 μm). the Biolog automatic microbiological analysis system tests 94 biochemical phenotypes of Clostridium sp.lz25 and identifies the species.71 carbon source utilization assays and 23 chemical sensitivity assays were analyzed in the 94 phenotypic tests.the important biochemical characteristics were determined by the microanalysis system GENIII microlatebiolog as shown in table 1, wherein Clostridium sp.lz25 is effective in using 9 carbon sources including dextrin, raffinose, D-sorbic acid, D-galacturonic acid, L-galacturonic acid lactone, D-glucuronic acid, glucuronamide, α -tetronic acid and acetoacetate, and 5 chemical species are sensitive including 1% NaCl and nalidixic acid, and sodium lactate, and 6% NaCl, and the most closely match the physiological characteristics of Clostridium strain 25.
Specifically, the obtained pure culture is inoculated on a BUG culture medium recommended by Biolog, a bacterial suspension is prepared in an IF-A inoculation liquid, the data of a microplate is read on a Biolog reading instrument, the reading is repeated for 3 times, and finally, the obtained pure culture is compared with a database for identification.
The identification method of the pyrene degradation strain Clostridium sp.LZ25 on the molecular level comprises the following steps: the 16S rDNA partial sequence of strain LZ25 was amplified using universal primers. The obtained fragments were sequenced, the 16S rDNA sequence was aligned by the NCBI database, and strain LZ25 was identified to clostridium on a molecular level.
The 16S rDNA PCR amplification conditions are as follows (the enzyme used is ExTaq): pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30S, annealing at 55 ℃ for 30S, extension at 72 ℃ for 90S, 30 cycles, extension at 72 ℃ for 5min, and agarose gel electrophoresis at 1.0% to confirm the amplified 16S rDNA fragment.
The invention also relates to application of the anaerobic strain Clostridium sp.strain LZ25 (hereinafter also referred to as LZ25) in the pyrene degradation treatment process, which comprises the following specific steps:
(1) strain Clostridium sp.z25 seed broth culture: adopting LB liquid culture medium as strain Clostridium sp.LZ25 seed liquid, taking strain Clostridium sp.LZ25 preserved on a flat plate, selecting colony from an anaerobic operation table by using an inoculating ring, inoculating into an anaerobic bottle containing LB liquid culture medium, carrying out shake culture, centrifuging thallus, and collecting seed liquid concentrate as degraded seed liquid;
(2) strain Clostridium sp.lz25 degrades pyrene: and (2) adding an inorganic salt culture medium into the pyrene-containing pollutants, inoculating the seed liquid obtained in the step (1), and performing degradation reaction under an anaerobic condition.
And (3) performing degradation reaction under the anaerobic condition in the step (2), wherein the seed liquid amount is 1-20%, and preferably 5-20%.
And (3) carrying out degradation reaction under anaerobic condition in the step (2), wherein the pH value of the reaction is 4.8-9.6, and the preferable pH value is 6.0-8.4.
The degradation reaction is carried out under the anaerobic condition in the step (2), and the reaction temperature is in the range of 22-50 ℃, and preferably 30-40 ℃.
And (3) carrying out degradation reaction under the anaerobic condition in the step (2), wherein the concentration range of reaction substrates pyrene is 50-200 mg/L, and preferably 50-100 mg/L.
According to an intermediate metabolite produced by pyrene degradation of a pyrene degrading strain Clostridium sp.LZ25 in the culture process, a metabolic pathway for the strain to degrade pyrene is presumed, and the method comprises the following specific steps:
(1) the pyrene-degrading strain Clostridium sp.LZ25 was cultured under optimum conditions for 40 days, and the amount of pyrene remaining in the culture was measured by liquid chromatography every 10 days.
(2) And selecting a system with relatively high pyrene degradation rate, and analyzing an intermediate metabolite in the pyrene degradation process through gas chromatography-mass spectrometry.
(3) Among the intermediate metabolites, representative organic substances were selected as substrates, the degradation rate thereof by the strains was examined, and the metabolites were analyzed.
(4) Comprehensively analyzing the intermediate metabolite, and analyzing the metabolic pathway of the strain for degrading pyrene according to the molecular structure.
The invention has the beneficial effects that: the anaerobic bacterium Clostridium sp.LZ25 can degrade polycyclic aromatic hydrocarbon, especially pyrene, under an anaerobic condition, has high degradation efficiency, is applied to the treatment of industrial sludge and sewage, and provides a feasible method for solving the problems of high cost and incomplete removal of polycyclic aromatic hydrocarbon of organic pollutants with high concentration. The method specifically comprises the following steps:
1. the strain has high efficiency of anaerobic degradation of pyrene, and the degradation rate of pyrene is obviously increased along with the increase of the inoculation amount.
2. The strain LZ25 has a wide temperature adaptation range when degrading pyrene, and can adapt to a wide pH range and a pyrene concentration range.
3. The invention has important application value for treating pollutants containing polycyclic aromatic hydrocarbon compounds.
Drawings
FIG. 1 is a schematic sp.LZ25 phylogenetic tree based on sequence homology of the 16S rDNA gene;
FIG. 2 pyrene degradation curve of strain LZ25 and its growth curve;
FIG. 3a pyrene degradation property of strain LZ25, effect of inoculation ratio on pyrene degradation by strain LZ 25;
FIG. 3b effect of temperature on pyrene degradation by strain LZ 25;
FIG. 3c effect of pH on pyrene degradation by strain LZ 25;
FIG. 3d Effect of initial substrate concentration on pyrene degradation by strain LZ 25;
FIG. 4 GC-MS analysis of pyrene degradation intermediates by Clostridium sp.LZ25;
fig. 5 Clostridium sp.lz25 vs. phenanthrene degradation curve;
fig. 6 GC-MS analysis of the intermediate product of phenanthrene degradation by Clostridium sp.lz 25;
FIG. 7 pathway of anaerobic degradation of pyrene by Clostridium sp.LZ25.
Detailed Description
The following detailed description of the embodiments of the present invention is provided, but it should be noted that the scope of the present invention is not limited by the embodiments, but is defined by the appended claims.
The experimental methods used in the following examples are all conventional methods without any special description, and the used experimental reagent consumables and the like can be purchased from commercial uses without any special description.
Example one
Separation and screening of pyrene-degrading strain Clostridium sp.lz 25:
(1) taking a petrochemical sludge flora containing polycyclic aromatic hydrocarbon as a screening target, and taking 10g of fresh sludge and 100mL of 0.85% physiological saline for oscillation for 20 min.
(2) To an anaerobic flask sterilized at 121 ℃ for 20min, a pyrene acetone solution having passed through a 0.22 μm organic filter was added, and the anaerobic flask was left to stand overnight with its opening for removing acetone.
(3) 50mL or 100mL of sterilized inorganic salt medium (pH 6.5-7.5) was added to the 100mL or 200mL anaerobic flask of step (2) in an anaerobic bench filled with nitrogen, and 0.1% resazurin solution as an oxygen indicator and cysteine as an oxygen scavenger were added, and finally a nitrogen seal was flushed. Wherein the inorganic salt culture medium: NaCl 24g/L, KCl0.7g/L, MgSO4·7H 20 0.7g/L、NH4Cl 1g/L、NaNO30.5g/L、KH2PO42g/L、Na2HPO43 g/L; wherein the concentration of pyrene is 20mg/L-200 mg/L. LB culture medium: NaCl 10g/L, peptone 10g/L and yeast powder 5 g/L. And adding agar 15-20 g/L into the LB culture medium to prepare an LB plate culture medium. The above culture medium is sterilized at 121 deg.C for 20min by high pressure steam sterilization.
(4) And (3) inoculating the suspension in the step (1) into an inorganic salt culture medium which takes pyrene (10mg/L) as a unique carbon source in the step (3) in an inoculation amount of 10%, and culturing for 7d at constant temperature of 30 ℃ and at an oscillation rate of 180 r/min. Then the culture medium is transferred into PAHs inorganic salt culture medium of 20mg/L again, and the culture is continued. This was repeated until the concentration of PAHs in the medium was 200 mg/L.
(5) Subjecting the last culture solution to 10-1To 10-9Gradient dilution and coating on LB plate culture medium, anaerobic bag culture, anaerobic culture at 30 deg.C for 3d, selecting out fast-growing and rich single colony, repeatedly drawing lines on the plate, and separating to obtain pure strain. To prevent pyrene from being photolyzed, the above steps were all performed in a dark room incubator.
(6) And (3) carrying out streak separation to obtain pure culture strains, selecting the strain with the highest efficiency of degrading pyrene, and naming the strain as the strain LZ 25.
(7) Inoculating the strain LZ25 into a liquid inorganic salt culture medium which takes 50mg/L pyrene as a unique carbon source, performing shake cultivation at 30 ℃ and 180r/min for 14 days, destructively sampling, extracting with dichloromethane, performing rotary evaporation, fixing the volume with methanol, and passing through a 0.22 mu m organic filter membrane, wherein the sample is used for detection and analysis of high performance liquid chromatography. The retention time of pyrene in the control culture solution without inoculation is about 7min, and after the strain LZ25 is inoculated and cultured for 14 days, the content of pyrene in the culture solution is obviously reduced.
Example two
Identification of pyrene-degrading strains:
the bacterial strain grows fast on an LB solid culture medium, yellow bacterial colonies with the diameter of 1mm can be formed at the temperature of 30 ℃ for 72 hours, the edges of the bacterial colonies are neat, the bacterial colonies are dull, prominent, sticky, moist, smooth and opaque, and the bacterial bodies are in a long rod shape (1.02 multiplied by 2.0 microns). Strain LZ25 was identified as the Clostridium genus based on its physiological and biochemical characteristics (table 1) and the 16S rRNA sequence. Finally, the base sequences of the strains with high homology were sequentially downloaded, and similarity analysis was performed using clustalZ, and a phylogenetic tree was constructed using the Neighbor-Joining method using MEGA 4 software (FIG. 2).
LZ25 can effectively utilize 9 carbon sources including dextrin, raffinose, D-sorbic acid, D-galacturonic acid, L-galacturonic acid lactone, D-glucuronic acid, glucuronamide, α -tetronic acid and acetoacetate while the chemosensitivity test is sensitive to 5 chemicals including 1% sodium lactate and nalidixic acid and can grow well at pH6, 1% NaCl and 4% NaCl the results of physiological and biochemical identification of strain LZ25 are most similar to those of Clostridium.
TABLE 1 determination of physiological and biochemical characteristics of Strain LZ25
Figure BDA0001793656410000061
EXAMPLE III
The degradation characteristic of pyrene degrading bacteria Clostridium sp.LZ25 comprises the following specific steps:
(1) strain LZ25 seed culture: strain LZ25 seed liquid adopts LB liquid culture medium, strain LZ25 preserved in the plate is taken, colony is picked out by inoculating loop in an anaerobic operating platform and is inoculated into an anaerobic bottle containing 50mL of LB liquid culture medium, shaking culture is carried out for 72 hours at 30 ℃,rotating the shaking table at 180r/min, reactivating the obtained bacterial liquid at a inoculation proportion of 10% for two times, centrifuging the thallus at 12000r/min for 10 min, collecting the liquid concentrate of the seed, and suspending in an inorganic salt culture medium to obtain OD600nm1.5 of bacterial suspension as degradation seed liquid;
(2) kinetics of pyrene degradation by strain LZ 25: inoculating seed solution into inorganic salt culture medium with pyrene initial concentration of 50mg/L according to 10% inoculation amount, shake culturing at 30 deg.C and 180r/min, destructively sampling at intervals, and determining OD600And residual concentration of pyrene.
(3) Effect of inoculum size and pH on pyrene degradation by strain LZ 25: inoculating seed liquid into an inorganic salt culture medium with the initial concentration of pyrene of 50mg/L according to the inoculation amounts of 1%, 5%, 10% and 20% respectively, and performing shake culture at 30 ℃ and 180 r/min; shaking culture at initial pH of 4.2, 6.0, 7.2, 8.4, 9.6, 30 deg.C and 180r/min, and determining the influence of inoculum size and pH on pyrene degradation by strain LZ 25.
(4) Effect of incubation temperature and initial concentration of substrate on pyrene degradation by strain LZ 25: inoculating seed liquid into an inorganic salt culture medium with the initial concentration of pyrene of 50mg/L according to the inoculation amount of 10%, and performing shake culture at 22 ℃, 30 ℃, 38 ℃, 50 ℃, pH 7.2 and 180r/min respectively; inoculating seed liquid into inorganic salt culture media with the initial concentrations of pyrene of 50mg/L, 100mg/L, 150mg/L and 200mg/L according to the inoculation amount of 10 percent, performing shake culture at 30 ℃ and 180r/min, and measuring the influence of the culture temperature and the initial concentration of a substrate on pyrene degradation of the strain LZ 25.
As can be seen from FIG. 2, when the strain LZ25 is inoculated into the culture medium for 5 days, pyrene degradation is started without obvious retardation; then the degradation rate is gradually increased, and after 30 days, the pyrene containing 50mg/L is degraded to about 28 mg/L. In addition, it can be seen from the figure that, as pyrene is degraded, the growth amount of the strain LZ25 begins to gradually increase after 5d of stagnation; when the degradation rate of pyrene reaches the maximum, the growth amount of the strain LZ25 also reaches the maximum; and the growth amount of the strain LZ25 begins to decrease at 30d-40 d. The results show that strain LZ25 can grow using pyrene.
As the inoculation amount is increased, the degradation rate of pyrene is obviously increased. When the inoculation amount is 1%, the degradation rate of pyrene at 30d is 21.2%; when the inoculation amount reaches 10%, the increasing speed of the pyrene degradation rate is slowed down, when the inoculation amount is 10%, the pyrene degradation rate is 45.3%, and when the inoculation amount is increased to 20%, the degradation rate reaches 50.0% (fig. 3 a). Clostridium sp.lz25 has substantially the same degradation rate for pyrene at pH 7.2 and 8.4; at 6.4, the degradation rate of pyrene is relatively inhibited; at pH 4.8 and 9.6, the degradation rate of pyrene was only 10% (FIG. 3 b). The strain LZ25 has a wide temperature adaptation range when degrading pyrene, and has good effect between 30 ℃ and 38 ℃; even at a culture temperature of 22 ℃, the degradation rate of pyrene could reach 29%. This result demonstrates that the degrading enzyme produced by strain LZ25 has a strong temperature flexibility (fig. 3 c). The degradation rate of pyrene by the strain LZ25 decreased with increasing substrate concentration. When the initial concentration of pyrene is 50mg/L, the degradation rate of pyrene at 30d is 45.3%; and when the initial concentration of pyrene is 200mg/L, the degradation rate of pyrene at 30d can still reach 14.2%, which shows that the strain has high efficiency of pyrene degradation (FIG. 3 d).
Example four
Degradation pathway of pyrene by pyrene degrading bacterium Clostridium sp.lz25:
qualitative analysis is carried out by GC-MS, and a series of intermediate metabolites in the anaerobic degradation process of pyrene by Clostridium sp.LZ25 are deeply researched. Chromatograms of the extracted samples showed that Clostridium sp.lz25 formed various metabolites during degradation of pyrene.
As can be seen from fig. 4, 7 species were found in the reaction system of Clostridium sp.lz25 at 10d, compared with the blank control. 13.426, 13.169, 11.411 and 13.350min are respectively pyrene (C16H10, CAS: 129-00-0), 4, 5-dihydropyrene (C16H12, CAS: 6628-98-4), phenanthrene (C14H10, CAS:85-01-8) and 1,1' - (1-butenylene) bis-benzene (C16H16, CAS: 1726-14-3) through mass spectrum comparison; 7.409, 10.779 and 23.203min are products of p-cresol (C7H8O, CAS: 106-44-5), protocatechenol (C7H6O4, CAS: 99-50-3) and benzoestrol (C20H26O2, CAS: 85-95-0) respectively after mass spectrum ratio silanization; the phenylestrol, p-cresol and protocatechol are assumed to be products produced in sequence after multiple metabolizations of 1,1' - (1-butenylene) biphenyl. The reaction system inoculated with Clostridium sp.LZ25 found two other new metabolites in the metabolite at 20d, and the chromatographic retention time was 6.575min and 7.993min, respectively, and the two metabolites were identified as products after silanization of phenol (C6H6O, CAS: 108-95-2) and phenethyl alcohol (C8H10O, CAS: 60-12-8) by mass spectrometry.
Since phenanthrene is found in the intermediate metabolites of pyrene, we used phenanthrene in the medium to further study the degradation pathway. Biodegradation experiments have shown that Clostridium sp.lz25 can degrade phenanthrene in addition to pyrene. LZ25 degraded 44.4% of 50mg/L phenanthrene after 40 days of culture (FIG. 5). The degradation time of phenanthrene is longer. After 30 days of culture, Clostridium sp.LZ25 degraded phenanthrene to only 24.5mg/L at an initial concentration of 50 mg/L. All of the above demonstrates that Clostridium sp.lz25 can utilize pyrene and phenanthrene as growth substrates.
In order to further research the degradation pathway of pyrene, the GC-MS technology is adopted to carry out qualitative analysis on the metabolic intermediates of phenanthrene. GC-MS analysis identified anaerobic metabolites of phenanthrene at day 20 (figure 6) compared to the phenanthrene blank, with derivatives detected after silanization of phenol and p-cresol at retention times of 6.682 and 7.402min in addition to the phenanthrene with a retention time of 11.405 min. Finally, silanized 2 '-hydroxy-4' -methylacetophenone (C9H10O2, CAS: 6921-64-8) was detected in the cultures of phenanthrene at a retention time of 15.365min on day 30 (FIG. 6). The above substances were confirmed by GC-MS analysis of standards, respectively.
From the analysis of the above intermediates, it was deduced that the pathway of Clostridium sp.lz25 anaerobic degradation of pyrene is shown in fig. 7. It can be seen that pyrene is reduced to 4, 5-dihydropyrene by adding two hydrogen atoms, and then c-c bond on saturated carbon atom is cleaved to generate phenanthrene. The hydrogenation reduction can be seen as an initial activation reaction of pyrene in the hypothetical pathway. Subsequently, phenanthrene is degraded to 1,1' - (1-butenylene) bis-benzene. 1,1' - (1-butenylene) biphenyl was converted into three products. One route is the conversion of 1,1' - (1-butenylene) biphenyl to phenylestrol by hydrolytic hydroxylation. The phenestrol is further converted into p-cresol, which is then hydroxylated to form p-hydroxybenzyl alcohol, which is then reacted with p-hydroxybenzaldehyde to form phenol. P-cresol may also be converted to protocatechuic acid by hydroxylation and carboxylation of the methyl group. 1,1'- (1-butenylene) bis-benzenes may also be converted to protocatechuic acid by hydrolysis and hydroxylation to form 2' -hydroxypropiophenone, re-methylation to form 1- (2-hydroxy-5-methylphenyl) -1-propanone, and re-carboxylation. Depending on the molecular structure, 1,1' - (1-butenylene) biphenyl may also produce a degradation product cinnamyl alcohol, which is further degraded to phenethyl alcohol, which is ultimately converted to phenol.

Claims (8)

1. An anaerobic strain is characterized by being classified and named as Clostridium sp.LZ25 with the preservation number as follows: CGMCCNO 15046.
2. The method of claim 1, wherein the 94 biochemical phenotypes of Clostridium sp.LZ25 are preliminarily identified by testing according to the Biolog automated microbiological analysis system, the obtained fragments are sequenced by amplifying the 16S rDNA partial sequence of the strain Clostridium sp.LZ25 using universal primers, the 16S rDNA sequence is aligned by NCBI database, and the strain Clostridium sp.LZ25 is identified to genus on a molecular level.
3. Use of the anaerobic strain Clostridium sp.lz25 according to claim 1 in pyrene biodegradation processes.
4. The use of the anaerobic strain Clostridium sp.lz25 according to claim 3 in the process of pyrene biodegradation, characterized by the specific steps of:
(1) strain Clostridium sp.lz25 seed broth culture: adopting LB liquid culture medium as strain Clostridium sp.LZ25 seed liquid, taking strain Clostridium sp.LZ25 preserved on a flat plate, picking bacterial colony in an anaerobic operating platform by using an inoculating loop, inoculating into an anaerobic bottle containing LB liquid culture medium, carrying out shake culture, centrifuging thallus, and collecting seed liquid concentrate as degraded seed liquid;
(2) strain Clostridium sp.lz25 degrades pyrene: and (2) adding an inorganic salt culture medium into the pyrene-containing pollutants, inoculating the seed liquid obtained in the step (1), and performing degradation reaction under an anaerobic condition.
5. The use of the anaerobic strain Clostridium sp.lz25 in the pyrene biodegradation process according to claim 4, wherein the amount of inoculated seed liquid in step (2) is 1-20%.
6. Use of the anaerobic strain Clostridium sp.lz25 according to claim 4 in the process of pyrene biodegradation, characterized in that the pH of the reaction in step (2) is 4.8-9.6.
7. Use of the anaerobic strain Clostridium sp.lz25 according to claim 4 in the process of pyrene biodegradation, characterized in that the reaction temperature in step (2) ranges between 22 ℃ and 50 ℃.
8. Use of the anaerobic strain Clostridium sp.LZ25 according to claim 4 in the biodegradation of pyrene, characterized in that the reaction substrate pyrene concentration in step (2) is in the range of 50mg/L to 200 mg/L.
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