Anaerobic strain for degrading PAH (polycyclic aromatic hydrocarbon)-pyrene as well as screening method and application of anaerobic strain
A screening method and polycyclic aromatic hydrocarbon technology, applied in the field of anaerobic bacterial strains that degrade polycyclic aromatic hydrocarbons-pyrene, can solve the problems of incomplete removal and high cost of polycyclic aromatic hydrocarbon removal, and achieve high degradation efficiency
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Embodiment 1
[0052] Isolation and screening of pyrene-degrading strain Clostridium sp.LZ25:
[0053] (1) Taking polycyclic aromatic hydrocarbon-containing petrochemical sludge flora as the screening target, take 10 g of fresh sludge and shake it in 100 mL of 0.85% normal saline for 20 min.
[0054] (2) Add the pyrene acetone solution that has passed through a 0.22 μm organic filter membrane into the anaerobic bottle that has been sterilized at 121°C for 20 minutes, and leave the anaerobic bottle open overnight to remove acetone.
[0055] (3) Add 50mL or 100mL of sterilized inorganic salt culture medium (pH=6.5-7.5) to the anaerobic bottle with a volume of 100mL or 200mL in step (2) in an anaerobic operating bench filled with nitrogen, and add 0.1 % resazurin solution as an oxygen indicator and cysteine as an oxygen scavenger, and finally flushed into nitrogen to seal. Inorganic salt medium: NaCl 24g / L, KCl0.7g / L, MgSO 4 ·7H 2 0 0.7g / L, NH 4 Cl 1g / L, NaNO 3 0.5g / L, KH 2 PO 4 2g / L...
Embodiment 2
[0061] Identification of pyrene-degrading strains:
[0062] The strain grows faster on LB solid medium, 30°C, 72h can form yellow colonies with a diameter of 1mm, the edges of the colonies are neat, dull, prominent, viscous, moist, smooth, opaque, and the cells are long rod-shaped (1.02 ×2.0 μm). Based on its physiological and biochemical characteristics (Table 1) and 16S rRNA sequence, strain LZ25 was identified as Clostridium genus. Finally, after downloading the base sequences of strains with high homology in sequence, similarity analysis was performed with clustaLZ, and a phylogenetic tree was constructed using the Neighbor-Joining method with MEGA 4 software ( figure 2 ).
[0063] Clostridium sp.LZ25 can effectively utilize 9 kinds of carbon sources, including dextrin, raffinose, D-sorbic acid, D-galacturonic acid, L-galacturonolactone, D-glucuronic acid, glucuronic acid amides, alpha-butyronic acid, and acetoacetate. At the same time, in the chemical sensitivity tes...
Embodiment 3
[0067] The degradation characteristics of pyrene-degrading bacteria Clostridium sp.LZ25, the specific steps are as follows:
[0068] (1) Bacterial strain LZ25 seed solution culture: Bacterial strain LZ25 seed solution adopts LB liquid medium, take the bacterial strain LZ25 preserved on the plate, use the inoculating loop to pick out colony and insert into the anaerobic bottle containing 50mL LB liquid medium in the anaerobic operation platform, Shake culture at 30°C for 72 hours, the shaker speed is 180r / min, and the obtained bacterial solution is activated twice at a seeding ratio of 10%, and the bacterial cell is centrifuged at 12000r / min for 10 minutes to collect the seed liquid concentrate, and resuspended in inorganic salt Medium, make OD 600nm =1.5 bacterial suspension as the degradation seed solution;
[0069] (2) Kinetics of bacterial strain LZ25 degrading pyrene: in the inorganic salt medium whose initial concentration of pyrene is 50mg / L, insert the seed solution ac...
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