Bacteria belonging to the genus cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment

Abstract

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 x 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 x 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.

FIG. 1.

Phylogenetic relationships based on 16S rDNA sequences of strains A5, E2, E3, and H4 and representative members of the γ subdivision of the Proteobacteria. Bootstrap values greater than 50% are indicated at the nodes. Scale bar = 0.01 substitution per site.

FIG. 2.

Phylogenetic relationships based on gyrB sequences of strains A5, E2, E3, and H4 and representative members of the γ subdivision of the Proteobacteria. Bootstrap values greater than 50% are indicated at the nodes. Scale bar = 0.05 substitution per site.

FIG. 3.

DGGE profiles of partial 16S rDNA fragments, showing major bacterial populations attached to the surfaces of oil-polluted grains of gravel. The lower panels show bands that were excised and used for the DNA sequencing analysis. Fresh seawater was continuously introduced into two beach-simulating tanks, and at zero time crude oil was poured into the tanks. In one tank no fertilizer was added (A), while in the other tank slow-release nitrogen and phosphorus fertilizers were added at zero time (B). Samples of the oil-polluted grains of gravel were collected on days −3, 0, 7, 21, 28, 42, and 56, as indicated at the top.

FIG. 4.

DGGE profiles of partial 16S rDNA fragments, showing major bacterial populations in seawater. The experimental conditions were the same as those described in the legend to Fig. 3. The lower panels show bands that were excised and used for the DNA sequencing analysis. (A) Samples from the fertilizer-free tank; (B) samples from the tank to which fertilizer was added. The seawater samples were collected on days −3, 0, 7, 21, 28, 42, and 56, as indicated at the top.

FIG. 5.

Propagation of Cycloclasticus on the surfaces of oil-polluted grains of gravel. The number of Cycloclasticus cells was estimated by q-cPCR. Symbols: □, cell number in the control tank; ▪, cell number in the tank to which fertilizer was added.