We found that bacteria closely related to Alcanivorax became a dominant bacterial population in petroleum-contaminated sea water when nitrogen and phosphorus nutrients were supplied in adequate quantity. The predominance of Alcanivorax bacteria was demonstrated under three experimental conditions: (i) in batch cultures of sea water containing heavy oil; (ii) in columns packed with oil-coated gravel undergoing a continuous sea water flow; and (iii) in a large-scale tidal flux reactor that mimics a beach undergoing tidal cycles with fresh sea water. These results suggest that bacteria related to Alcanivorax are major players in the bioremediation of oil-contaminated marine environments.

To identify the bacteria that play a major role in the aerobic degradation of petroleum polynuclear aromatic hydrocarbons (PAHs) in a marine environment, bacteria were enriched from seawater by using 2-methylnaphthalene, phenanthrene, or anthracene as a carbon and energy source. We found that members of the genus Cycloclasticus became predominant in the enrichment cultures. The Cycloclasticus strains isolated in this study could grow on crude oil and degraded PAH components of crude oil, including unsubstituted and substituted naphthalenes, dibenzothiophenes, phenanthrenes, and fluorenes. To deduce the role of Cycloclasticus strains in a coastal zone oil spill, propagation of this bacterial group on oil-coated grains of gravel immersed in seawater was investigated in beach-simulating tanks that were 1 m wide by 1.5 m long by 1 m high. The tanks were two-thirds filled with gravel, and seawater was continuously introduced into the tanks; the water level was varied between 30 cm above and 30 cm below the surface of the gravel layer to simulate a 12-h tidal cycle. The number of Cycloclasticus cells associated with the grains was on the order of 10 3 cells/g of grains before crude oil was added to the tanks and increased to 3 ؋ 10 6 cells/g of grains after crude oil was added. The number increased further after 14 days to 10 8 cells/g of grains when nitrogen and phosphorus fertilizers were added, while the number remained 3 ؋ 10 6 cells/g of grains when no fertilizers were added. PAH degradation proceeded parallel with the growth of Cycloclasticus cells on the surfaces of the oil-polluted grains of gravel. These observations suggest that bacteria belonging to the genus Cycloclasticus play an important role in the degradation of petroleum PAHs in a marine environment.An oil spill is one of the most serious disasters that can occur in a marine environment (22,23). In the early stage of an oil spill, the light fraction of the oil evaporates, while the heavier fraction is slowly removed by photooxidation and biodegradation (11). Microorganisms, especially bacteria, play an important role in the biodegradation of the spilled oil (28). However, the growth of oil-degrading bacteria in seawater and the resulting biodegradation of oil in seawater are limited by nutritional requirements (2, 3). Addition of nitrogen and phosphorus fertilizers has been shown to enhance the biodegradation of oil released in a marine environment (21,38,43).Oil is a complex mixture made up of hundreds of compounds, and these compounds are classified into four groups, namely, saturates, aromatics, resins, and asphaltenes (12). Aromatics are the second most abundant hydrocarbons in crude oil. Benzene, naphthalene, and phenanthrene and their alkylsubstituted derivatives represent typical aromatics (47). Although the biodegradation of such simple aromatics as benzene, toluenes, xylenes, naphthalene, and phenanthrene has been extensively characterized (19), the biodegradation of alkyl-substituted polynuclear aromatic hydrocarbons (PAHs) has scarcely bee...

In January 1997, the tanker Nakhodka sank in the Japan Sea, and more than 5000 tons of heavy oil leaked. The released oil contaminated more than 500 km of the coastline, and some still remained even by June 1999. To investigate the long-term influence of the Nakhodka oil spill on marine bacterial populations, sea water and residual oil were sampled from the oil-contaminated zones 10, 18, 22 and 29 months after the accident, and the bacterial populations in these samples were analysed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments. The dominant DGGE bands were sequenced, and the sequences were compared with those in DNA sequence libraries. Most of the bacteria in the sea water samples were classified as the Cytophaga-Flavobacterium-Bacteroides phylum, alpha-Proteobacteria or cyanobacteria. The bacteria detected in the oil paste samples were different from those detected in the sea water samples; they were types related to hydrocarbon degraders, exemplified by strains closely related to Sphingomonas subarctica and Alcanivorax borkumensis. The sizes of the major bacterial populations in the oil paste samples ranged from 3.4 x 10(5) to 1.6 x 10(6) bacteria per gram of oil paste, these low numbers explaining the slow rate of natural attenuation.

The genus Alcanivorax comprises diverse hydrocarbon-degrading marine bacteria. Novel 16S rRNA-targeted oligonucleotide DNA probes (ALV735 and ALV735-b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single-mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil-contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4',6-diamidino-2-phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon-degrading microbial consortia.