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Isolation and Identification of Pyrene-Degrading Strain SE12 and Its Degrading Effect

Author: ZhangQiaoQiao
Tutor: RanZuo
School: Nanjing Agricultural College
Course: Environmental Engineering
Keywords: Pyrene Microbial degradation Mycobacterium SE12 Remediation of contaminated soils Activity
CLC: X172
Type: Master's thesis
Year: 2010
Downloads: 13
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Polycyclic aromatic hydrocarbons (PAHs) represent an important class of environmental pollutants, of which most are known to be carcinogenic, teratogenic and mutagenic. Pyrene, which has four fused aromatic rings, has been detected in environmental samples and used as an indicator for PAHs pollution monitoring and usually as the representative of PAHs with more than four aromatic rings. Since high PAHs contents have been reported in some soil and sediment environments of China, we are concerned that the high potential of PAHs threatens human health and environmental quality. However, many of the remediation techniques for PAHs in soil or sediment are still in the investigational stages and have been applied only in the laboratory or on a small scale. Microbiological remediation of soil or sediment organic pollutants generally refers to the breakdown of organic compounds by living microbes eventually resulting in the formation of carbon dioxide and water or methane. Microbiological methods have advantages over physical and chemical methods in land requirement, overall costs and lasting effect. Isolation and screening of high efficient strain are still the essential work for remediation of soil and sediment PAHs pollution. Many previous works showed a variety of microbes can degradate three or less than three ring PAHs like phenanthrene, but few works have been done on microbes that can effectively break down four or more than four ring PAHs and their bioremediation. This paper attempts to isolate high-effective pyrene-degrading bacterial strains from contaminated soil around a coking plant in Woniushan, Xuzhou, and to evaluate the effeciecies of pyrene-degrading strains in remediation of soil pyrene pollution. The main results were presented as following.1. Strain SE12 that used pyrene as sole carbon and energy sources was isolated from the contaminated soil by plate sublimation method. The strain was identified by morphological, biochemical and 16S rDNA analysis, as a Mycobacterium sp. Strain (GenBank accession number:GU134623), which had 98% identity of the rapid-growth nonpathogen strain M. austroafricanum ATCC 33464. By using primer pairs of nidAF/ nidAR and nidBF/ nidBR for amplification of the ring-hydroxylating dioxygenase gene, results showed that SE12 had dioxygenase genes, which encode the large and small subunits of dioxygenase. Sequence analysis showed that the genes were highly homologous to the dioxygenase genes of pyrene-degrading Mycobacteria species (the GenBank accession number of SE12 nidA and nidB are GU586859 and GU586860).2. SE12 had excellent effect in degradation of pyrene in liquid media. The results showed that when the initial pyrene concentration were 50,100 and 200 mg L-1, the pyrene degradation rates were 86.4%,96.4% and 79.6%. The pyrene was almost completely degraded by SE12 when the initial concentration of pyrene was 100 mg L-1. The degradation rate of pyrene has been reduced when the initial concentration of pyrene lower or higher than 100 mg L-1. The 100 mg L-1 pyrene could be degraded by SE12 in the range of pH 5-10. However, pH 9 was the best point, at which the degradation rate of pyrene was 97.1%. The cell number of strain SE12 was more than 1×107 cell mL-1 when temperatur was in the range of 25-40℃. The cell number of strain SE12 was in accord with the pyrene degradation rate. The pyrene degradation rate markedly decreased when the system temperature lower than 25℃or higher than 40℃. In addition, the cell number of strain SE12 was more affected than the rate of pyrene degradation when different metabolites were added in system.3. Strain SE12 had strong ability to degrade pyrene in soil environment. When soils were amended with 10mg·kg-1 and 200 mg·kg-1 of pyrene and cultured for 28 d, soil pyrene contents were decreased by 65.1% and 96.7%, respectively, for pre-sterilized soil, and by 76% and 98.7%, respectively, for non-sterilized soil.4. The responses of different soil enzymes to addition of pyrene and SE12 in soil were different. Soils were sampled at 0,3,7,14, and 28 d after pyrene and SE12 were added to soil. Activities of dehydrogenase, polyphenol oxidase and saccharase in soil were continually increased, while activities of catalase were not siginificantly changed. Activities of urease increased in the first two weeks and then decreased to the original state.5. After pyrene and SE12 were added to soil, protein-like substances in soil water dissolved organic matter (DOM) were increased. Results from three-dimensional excitation emission matrix fluorescence spectroscopy analysisy showed protein-like fluorescence peaks were significantly increased in DOM samples after pyrene and SE12 were added to soil, suggesting that there were high microbiological activity in soil.In conclusion, this study showed that strain SE12 was effective to degrade pyrene both in liquid media and actual soil environment and had an engineering potential to be used in bioremediation of polycyclic aromatic hydrocarbons contaminated soil. Furthor studies should be laid on for the performance of SE12 at in situ bioremediation of polluted fields.

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