ISOLATION AND CHARACTERIZATION OF POLYCYCLIC AROMATIC HYDROCARBON-DEGRADING MICROORGANISMS UNDER METHANOGENIC CONDITIONS

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are among the most widely distributed organic contaminants in aquatic sediments due to their presence in coal and petroleum. While it has been demonstrated that PAHs are degraded under anaerobic conditions, little is known about the microorganisms responsible for PAH degradation. This study demonstrates not only the first isolations of naphthalene (NAP)- and phenanthrene (PHE)-degrading microorganisms under methanogenic conditions by utilizing modified plating methods but also the first identification and isolation of a fermentative bacterium responsible for initiating a syntrophic PHE-degradation. Molecular characterization of PAH-degrading methanogenic cultures via comparative 16S rDNA sequence analysis was employed to monitor the microbial community structure and consequently to design isolation strategies for the possible microbial species responsible for PAH-degradation. To isolate PAH-degrading microbes under anaerobic conditions, a modified plating method was first developed for detecting microorganisms degrading solid PAHs on the agar-overlay plate. It was also verified that this method was not only applicable for the isolation of both aerobic and anaerobic PAH-degrading microorganisms but also effective to solve problems existing with other previous isolation methods. By employing the modified plating method, PHE-degrading microorganisms under methanogenic conditions were successfully isolated from the enrichment cultures. The degradation of PHE was partially inhibited by 2-bromoethanesulfonic acid; however, no 14CH4 was detected when [9-14C] PHE was employed, indicating partial mineralization of PHE. One species of bacterium was isolated and identified as an initial microbial catalyst for PHE-degradation. NAP-degrading microorganisms under methanogenic conditions were also isolated by employing an agar-overlay containing evenly dispersed fine particles of NAP. One species of the bacteria was identified to be the same microorganism as a fermentative bacterium initiating a syntrophic PHE-degradation, and the other one showed a syntrophic relationship with methanogen species. The results presented here will likely contribute to the development of the isolation techniques and the identification of microbial consortia for the biodegradation of PAHs under anaerobic conditions.