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Applied and Environmental Microbiology, October 2000, p. 4205-4211, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Rapid Mineralization of Benzo[a]pyrene by a Microbial Consortium Growing on Diesel Fuel

Robert A. Kanaly,1,2,* Richard Bartha,1 Kazuya Watanabe,2 and Shigeaki Harayama2

Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231,1 and Marine Biotechnology Institute, Kamaishi Laboratories, 3-75-1 Heita, Kamaishi City, Iwate 026-0001, Japan2

Received 6 April 2000/Accepted 6 July 2000

A microbial consortium which rapidly mineralized the environmentally persistent pollutant benzo[a]pyrene was recovered from soil. The consortium cometabolically converted [7-14C]benzo[a]pyrene to 14CO2 when it was grown on diesel fuel, and the extent of benzo[a]pyrene mineralization was dependent on both diesel fuel and benzo[a]pyrene concentrations. Addition of diesel fuel at concentrations ranging from 0.007 to 0.2% (wt/vol) stimulated the mineralization of 10 mg of benzo[a]pyrene per liter 33 to 65% during a 2-week incubation period. When the benzo[a]pyrene concentration was 10 to 100 mg liter-1 and the diesel fuel concentration was 0.1% (wt/vol), an inoculum containing 1 mg of cell protein per liter (small inoculum) resulted in mineralization of up to 17.2 mg of benzo[a]pyrene per liter in 16 days. This corresponded to 35% of the added radiolabel when the concentration of benzo[a]pyrene was 50 mg liter-1. A radiocarbon mass balance analysis recovered 25% of the added benzo[a]pyrene solubilized in the culture suspension prior to mineralization. Populations growing on diesel fuel most likely promoted emulsification of benzo[a]pyrene through the production of surface-active compounds. The consortium was also analyzed by PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments, and 12 dominant bands, representing different sequence types, were detected during a 19-day incubation period. The onset of benzo[a]pyrene mineralization was compared to changes in the consortium community structure and was found to correlate with the emergence of at least four sequence types. DNA from 10 sequence types were successfully purified and sequenced, and that data revealed that eight of the consortium members were related to the class Proteobacteria but that the consortium also included members which were related to the genera Mycobacterium and Sphingobacterium.

* Corresponding author. Mailing address: Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi City, Iwate, 026-0001, Japan. Phone: 81-193-26-5781. Fax: 81-193-26-6584. E-mail: robert.kanaly{at}kamaishi.mbio.co.jp.

Applied and Environmental Microbiology, October 2000, p. 4205-4211, Vol. 66, No. 10
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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