Novel Metabolites of Anaerobic Naphthalene Biodegradation by Pure Cultures

We have previously reported on anaerobic pure cultures that degrade naphthalene in the strict absence of oxygen. One of these pure cultures NAP-3 was phylogenetically most closely related to Pseudomonas stutzeri based on 16S rDNA sequences. Further studies in our laboratory have shown that the degradation of naphthalene was slow or nearly absent when volatile fatty acids (VFAs) were not present. To investigate this further, we analyzed NAP-3 during incubations with acetate and found that it stimulates naphthalene transformation. Naphthalene significantly increased denitrification in nearly exact stiochiometric balances of nitrate and nitrite as would be expected based on the metabolism. NAP-3 was sensitive to the amount of naphthalene in the culture; with naphthalene removal rates higher when present at 20mg/l compared to 40mg/l. Investigation of the culture supernatant by GC/MS showed the transient production and consumption of the nitrogen-containing bicyclic indole. Production of indole was repeatable and found to be affected by naphthalene concentration; with the 20mg/l culture producing the most indole. The production of indole was strictly biotic (it was not formed in either killed or blank controls). It is not immediately apparent which metabolic pathway could form indole from naphthalene, but it possibly could involve the biotic incorporation of either nitrate or a reduced metabolite of nitrate into the nitrogencontaining ring of indole. To date, the only published metabolic pathways for anaerobic PAH biodegradation is ring hydroxylation or carboxylation by sulfate-reducers. These results show that other metabolic pathways exist for denitrifiers, and may require stimulation of the organisms by more-readily utilizable VFA substrates. INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are of concern in the environment because of their persistence and their potential carcinogenic effects. PAHs are introduced into the environment through natural and anthropogenic processes causing them to be ubiquitous in the environment. Over the past 20 years, numerous studies have reported the capacity of various bacteria, fungi and algae to degrade PAHs (See Makkar and Rockne, 2003 for a review). However, few studies have focused on the contribution of individual microorganisms in reducing the toxicity of PAH compounds. With recent results clearly demonstrating some bicyclics and PAHs can be degraded without oxygen, further progress in understanding this process will be achieved by the identification of pure cultures of anaerobic PAH degrading bacteria. Many pure cultures of denitrifying and sulfate reducing bacteria able to degrade mono-aromatics like toluene have been determined (Karthikeyan, 2001). Anaerobic degradation of monoaromatic hydrocarbons proceeds in every case investigated through the benzoyl-coenzyme A (benzoyl-CoA) pathway and differs only with respect to the peripheral degradation pathways (Johann et al, 1999). In contrast to the degradation of monoaromatic compounds, much less information exists on the anaerobic degradation of polycyclic aromatic hydrocarbons. In one study, naphthalene was microbially transformed in sulfate-reducing laboratory microcosms established under strictly anaerobic conditions with the formation of naphthol as a potential metabolic intermediate, suggesting that hydroxylation may be a first step in the sulfate-mediated transformation of naphthalene (Bedessem and Colberg, 1997). Earlier studies were done on naphthalene degradation by sulfate-reducing bacterial cultures with a marine enrichment culture from harbor sediment (Zhang and Young, 1997, Zhang and Young, 2000) and a freshwater culture enriched from a tar-oil contaminated aquifer (Annweiler and Meckenstock, 2000, Meckenstock, 2000, Morasch and Meckenstock, 2001). These studies showed that 2-naphthoic acid was an intermediate formed by the incorporation of bicarbonate into the carboxy group, suggesting that carboxylation is the initial step for degradation of naphthalene by sulfatereducers (Zhang and young, 2000, Makkar and Rockne, 2003). It has been also proposed that degradation of PAHs (mainly bicyclic) compounds goes through carboxylation and then through ring saturation with 2-naphthoic acid is the central intermediate (Annweiler and Meckenstock, 2002). FIGURE 1. Proposed pathway for naphthalene transformation by sulfate-reducing bacteria. We previously reported on the isolation of pure cultures of anaerobic PAHdegrading bacteria derived from a denitrifying enrichment culture (Rockne and Strand, 1999). The pure culture was phylogenetically most closely related to Pseudomonas stutzeri based on 16S rDNA sequences. We subsequently found that the degradation of naphthalene by NAP-3 (the pseudomonad) was slow or nearly absent when volatile fatty acids (VFAs) were not present. To investigate this further, studies were performed to demonstrate the biotransformation of naphthalene by NAP-3 during incubations with acetate present. In this report we also studied in detail the nitrate transformation kinetics by Nap 3 culture with and without naphthalene. MATERIALS AND METHODS Bacterial strain. The microorganism used (NAP-3) was a pure culture previously shown to degrade naphthalene with stiochiometric amounts of nitrate reduction under strictly anaerobic conditions (Rockne et al, 2000). NAP-3 was phylogenetically most closely related to Pseudomonas stutzeri based on 16S rDNA sequences. Naphthalene COOH