nitrate-reducing and denitrifying pure cultures. In-Situ and On-Site Bioremediation. 5(8):271-276. NAPHTHALENE DEGRADATION AND MINERALIZATION BY NITRATE-REDUCING AND DENITRIFYING PURE CULTURES

Although most PAHs with fewer than five rings are known to be biodegraded under aerobic conditions, most contaminated sediments and soils are anaerobic. In these environments, aerobic bioremediation may be difficult to implement because of the problems associated with oxygen delivery to the subsurface. 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. We attempted to isolate pure bacterial cultures from a denitrifying enrichment that anaerobically degrades phenanthrene, naphthalene, and biphenyl with stoichiometric nitrate reduction. After enrichment and screening, four pure cultures were obtained. The isolates were assayed for the ability to degrade naphthalene or phenanthrene as the sole source of carbon and energy in the presence of nitrate. Two pure cultures demonstrated unambiguous naphthalene biodegradation ability, designated NAP-3-1 and NAP-4. Both NAP-4 and NAP-31 transformed naphthalene, and the transformation was nitrate dependent. No significant removal of naphthalene occurred in nitrate-limited incubations or in cell-free controls. Both cultures partially mineralized naphthalene, representing 12-15% of the initial added C-labeled naphthalene. Isolate NAP-3-1 was a denitrifier, as shown by gas production in a denitrification assay. In contrast, NAP-4 did not produce gas, but did produce significant amounts of nitrite. The complete 16s rDNAs of NAP-3-1 and NAP-4 were sequenced and compared to other PAHand non-PAH degrading bacteria. NAP-4 was phylogenetically closely related to Vibrio spp. and NAP-3-1 was phylogenetically closely related to Pseudomonas spp., results which suggest that anaerobic bicyclic degradation ability may be more widely spread within the proteobacteria. INTRODUCTION Over the last decade our understanding of anaerobic aromatic biodegradation has increased greatly. With the isolation of denitrifying and sulfate reducing pure cultures able to degrade toluene, several biodegradation pathways and phylogenetic relationships between strains have been determined. These 192 Bioremediation Technologies for Polycyclic Aromatic Hydrocarbon Compounds studies led to the finding that this metabolic ability is relatively widespread. In contrast, until recently anaerobic biodegradation of unsubstituted aromatics like benzene and polycyclic aromatic hydrocarbons (PAHs) has not been widely reported (with a few exceptions, e.g. Mihelcic and Luthy, 1988). In fact, anaerobic biodegradation of these compounds was thought unlikely to be biochemically possible due to the lack of ring constituents such as the methyl group on toluene (Evans and Fuchs, 1988). In the last three years however, anaerobic biodegradation of bicyclics and PAHs such as biphenyl, naphthalene, phenanthrene, fluorene, and fluoranthene have been conclusively demonstrated in marine sulfate-reducing and denitrifying enrichment cultures derived from Puget Sound, WA (Rockne and Strand, 1998), Arthur Kill, NJ (Zhang and Young, 1997), and San Diego Harbor, CA (Coates et al., 1997). At present it is unknown why this activity is seemingly more prevalent in marine environments. Although a pathway for initial PAH ring oxidation has been found using a mixed sulfate-reducing enrichment culture (Zhang and Young, 1997), detailed phylogenetic analysis and further biochemical pathway elucidation would be greatly enhanced by studies with pure cultures. In the work reported here, we give an overview of the isolation of pure cultures of anaerobic PAH-degrading bacteria derived from a denitrifying enrichment culture previously demonstrated to degrade PAHs anaerobically (Rockne and Strand, 1998). MATERIALS AND METHODS The source of inoculum for the isolation procedures was a highly enriched denitrifying culture previously shown to degrade naphthalene, biphenyl, and phenanthrene with near stoichiometric amounts of nitrate reduction under strictly anaerobic conditions (Rockne and Strand, 1998). The culture was initially enriched on a mixture of the PAHs in a fluidized bed reactor (FBR), but was subcultured on individual PAHs in batch culture studies demonstrating stoichiometric biodegradation and mineralization of the PAHs. A schematic of the entire isolation procedure used in this work is shown in Figure 1. A complete description of the isolation procedure is found in Rockne et al. (1999). Briefly, cells were first transferred to agar plates containing nitrate-amended R2A denitrifying artificial seawater (ASW) media. R2A has a variety of electron donors for selection of heterotrophic bacteria. Cells from individual colonies on the R2A plates were transferred to liquid media with individual PAHs. Liquid cultures demonstrating growth (by turbidity) were transferred to agar shake (AS) tubes fed PAH by diffusion through the agar from a hexadecane phase as described previously (Rockne et al., 1999). Individual colonies from the AS tubes were transferred to liquid culture containing nitrate and PAHs. Cultures showing growth with PAH and nitrate transformation were plated twice on R2A agar and the resulting colonies were assayed for PAH degradation, denitrification, and mineralization as described previously (Rockne et al., 1999). Colonies of selected strains grown anaerobically on R2A plates were harvested and genomic DNA was isolated as described previously (Rockne et al.,