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Applied and Environmental Microbiology, April 2000, p. 1595-1601, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Anaerobic Naphthalene Degradation by Microbial Pure Cultures under Nitrate-Reducing Conditions

Karl J. Rockne,1,dagger Joanne C. Chee-Sanford,2,* Robert A. Sanford,2,Dagger Brian P. Hedlund,2 James T. Staley,2 and Stuart E. Strand3

Department of Civil Engineering,1 Department of Microbiology,2 and College of Forest Resources,3 University of Washington, Seattle, Washington 98195

Received 4 October 1999/Accepted 20 January 2000

Pure bacterial cultures were isolated from a highly enriched denitrifying consortium previously shown to anaerobically biodegrade naphthalene. The isolates were screened for the ability to grow anaerobically in liquid culture with naphthalene as the sole source of carbon and energy in the presence of nitrate. Three naphthalene-degrading pure cultures were obtained, designated NAP-3-1, NAP-3-2, and NAP-4. Isolate NAP-3-1 tested positive for denitrification using a standard denitrification assay. Neither isolate NAP-3-2 nor isolate NAP-4 produced gas in the assay, but both consumed nitrate and NAP-4 produced significant amounts of nitrite. Isolates NAP-4 and NAP-3-1 transformed 70 to 90% of added naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred under nitrate-limited conditions or in cell-free controls. Both cultures exhibited partial mineralization of naphthalene, representing 7 to 20% of the initial added 14C-labeled naphthalene. After 57 days of incubation, the largest fraction of the radiolabel in both cultures was recovered in the cell mass (30 to 50%), with minor amounts recovered as unknown soluble metabolites. Nitrate consumption, along with the results from the 14C radiolabel study, are consistent with the oxidation of naphthalene coupled to denitrification for NAP-3-1 and nitrate reduction to nitrite for NAP-4. Phylogenetic analyses based on 16S ribosomal DNA sequences of NAP-3-1 showed that it was closely related to Pseudomonas stutzeri and that NAP-4 was closely related to Vibrio pelagius. This is the first report we know of that demonstrates nitrate-dependent anaerobic degradation and mineralization of naphthalene by pure cultures.

* Corresponding author. Mailing address: Department of Animal Sciences, 454 ASL MC-630, University of Illinois, Urbana, IL 61801. Phone: (217) 333-8809 voice, (217) 333-8804 Fax. E-mail: cheesanf{at}ux1.cso.uiuc.edu.

dagger Present address: Department of Chemical and Biochemical Engineering, Rutgers, The State University of New Jersey, Piscataway, N.J.

Dagger Present address: Department of Civil and Environmental Engineering, University of Illinois, Urbana, Ill.

Applied and Environmental Microbiology, April 2000, p. 1595-1601, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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