| Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, some of which are highly carcinogenic, genotoxic, and a threat to public health. The major sources of PAHs in the environment are the combustion of organic matter and the processing and use of fossil fuels. Chemical-physical as well as biological methods are used to remove PAHs, Above all biological methods are favored because of good results and low costs. In our previous research work, we have obtained a set of dominant bacteria from sludge contaminated by coke plant wastewater which could degrade PAHs effectively. On that basis, we further researched biodegradation and enzymatic degradation characteristics for Flavobacterium sp CN4 and Bacillus CN2, which were isolated from coke plant sludge. The results provide theoretical basis for understanding PAHs biodegradation mechanism.Using beef extract peptone medium to culture two strains which were maintained on beef-peptone agar slants, and the bacteria-suspended buffer of two strains were respectively prepared. Two bacteria also could grow with pyrene or glucose as alternative carbon sources. And pyrene-grown strains produced higher levels of PAHs degradation activity than glucose-grown bacteria. After 5-d degradation, the removal efficiency of anthracene by CN4 which were grown with pyrene as the sole carbon source for 48-h was 1.50-fold more than the bacteria were cultured with glucose as the sole carbon source. And 96-h incubation CN2 by using pyrene and glucose as carbon source, the removal efficiency of anthracene within 72-h by CN2 was 1.80-fold more than the bacteria grown on glucose. The main influence factors of PAHs biodegradation and kinetic characteristics in aqueous were investigated by using UV spectrophotometer. The results showed CN4 and CN2 had higher activity for degrading anthracene, phenanthrene, pyrene at pH 5-7. At pH 5, the removal efficiency of anthracene, phenanthrene, pyrene by CN4 was 27.6%,33.5%,26.4% within 24h, respectively,. At pH 6, the removal efficiency of anthracene, phenanthrene, pyrene by CN2 was 33.5%,46.5%,36.5% within 24h, respectively. Biodegradation was inhibited When PAHs concentrations was over 200mg/L and cell concentration was 1.0×107CFUs·mL-1.Phenanthrene metabolites by Flavobacterium sp CN4 were assayed by using Purge and Trap combing with gas chromatography-mass spectrometry (GC-MS). Metabolism of phenanthrene by Bacillus CN2 were analyzed using liquid-liquid extraction combing with GC-MS and Fourier transform infrared spectroscopy (FTIR). In phenathrene degradation by CN4, the major metabolites include Coumarin, Phthalates, cinnamate, hydroxy-naphthoic acid, protocatechuic acid, phenanthrenedihydrodiol, phenanthrenediol. And the main metabolites were Phthalates and phenanthrenequinone when phenathrene was degraded by CN2. The change of functional groups between biodegradation metabolites and phenanthrene was given by comparison of FTIR spectra of phenanthrene with metabolites. Degradation pathway was proposed by identifying metabolites of phenanthrene. Degradation of phenanthrene by CN4 and CN2 were both initiated by dioxygenation, and then ring cleavages were happened to produce carboxylic acid. However, the metabolites of phenathrne by two strains were not quite the same. This indicated two strains had different enzyme system.To detect active enzymes which were produced by two strains, salting out method and ultrasonication were respectively used to obtained extracellular enzyme and endoenzyme, and PAH enzyme degradation experiments were executed. The results showed Coarse endoenzyme excreted from two strains could degrade pyrene effectively. Within 30 minutes, the removal rates of the pyrene were 26.5% and 20.8% by endoenzyme produced from CN4 and CN2, respectively. However, the extracellular enzyme didn't show degradation activity toward pyrene within 6 hours. These results showed endoenzymes excreted from two strains were active enzyme. To obtained endoezyme, the optimum ultrasonication conditions were as follows:CN4 or CN2 bacterial suspensions were subjected to sonication for 15 min on ice with a cell ultrasonifier, the amplitude 260W(280W at most) and 30-s pulse intervals. The lysate was centrifuged for 20min at 11000 r·min-1 to remove cell debris.Enzymatic-degradation kinetic characteristics of PAHs in aqueous were investigated. At 32℃and pH 6, the endoenzyme was the most active. The coarse endoenzyme of two strains had higher activity for degrading anthracene, phenanthrene, pyrene in weak acid medium at a temperature between 30℃and 35℃. The endoenzyme enzyme lost degrading activity when temperatures exceed 50℃. Enzymatic degradation reaction rate keep linear relation with PAH concentrations when substrate concentrations were not more than 200mg/L. The average initial rates were increasing with PAH initial concentration, but the average initial rates were decreasing with time. The apparent reaction orders were roughly the same when enzymatic degradation time was different.Various metal ions were investigated as additives which could potentially enhance enzyme activity. It was found that Ca2+ could markedly activate the coarse enzymes. As 0.5mmol/L Ca2+ was added, the removal rates of anthracene, phenanthrene and pyrene by coarse endoenzyme of CN4 were improved by 1.3,1.9 and 1.8 times, respectively. And the removal rates of anthracene, phenanthrene and pyrene by coarse endoenzyme of CN2 were improved by 1.4,1.6 and 1.4 times, respectively. The mechanism of this result is that metal ion provide a bridge for enzyme to substrate, Which bind enzyme to form ion-enzyme complex. Then bind substrate to form enzyme-ion-substrate complex.
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