Indole production by Pseudomonas stutzeri strain NAP-3 during anaerobic naphthalene biodegradation in the presence of dimethyl formamide

Abstract

We previously reported on the denitrifier Pseudomonas stutzeri strain NAP-3 that degrades naphthalene without oxygen. In our present studies, naphthalene biodegradation by NAP-3 was slow or nearly absent when acetate was not present, suggesting a stimulating effect by the non-fermentable substrate. NAP-3 was sensitive to the amount of naphthalene in the culture; with naphthalene removal and nitrate utilization rates higher when present at 20 mg L^{− 1} compared to 40 mg L^{− 1}. Electron equivalents represented by transformation of electron donor could be met assuming nitrate reduction to nitrite, except for the incubation with 40 mg L^{− 1} naphthalene which required complete denitrification. Transient production of the nitrogen-containing bicyclic indole was found by gas chromatograph mass spectrophotometry (GC/MS). Indole production was repeatable and affected by naphthalene concentration; with the 20 mg L^{− 1} culture producing the most indole. The production of indole was strictly biotic and required the presence of both naphthalene and the commonly-used solubilizing agent N, N dimethyl formamide (DMF). The source of nitrogen in indole was confirmed to be DMF through a series of growth experiments with NAP-3. No indole was produced in incubations fed naphthalene plus nitrate, nitrite or ammonia, either alone or in combination, when DMF was absent. Further, no production of indole was observed when heptamethyl nonane was used as an alternative solubilizing agent. We speculate that indole production proceeds through a pathway similar to the chorismic acid pathway for indole synthesis with incorporation of nitrogen from DMF. No other potential compounds of the chorismic pathway were found by GC/MS when naphthalene was the sole carbon source. NAP-3 grew statistically significantly on anthranilate, but not using salicylate or indole as the sole carbon source.

Keywords:

• PAH
• polycyclic aromatic hydrocarbon
• nitrate reduction
• denitrification
• dimethyl formamide
• indole
• naphthalene
• Pseudomonas stutzeri

Acknowledgment

This project was funded by National Science Foundation grant BES-0348512. Additional funding came from NOAA/UNH Cooperative Institute for Coastal and Estuarine Environmental Technology grant number NA97OR0338. We also thank Dr. Randhir Makkar (currently at the Medical University of South Carolina) for assistance in the laboratory.