Rhodanobacter sp. strain BPC1 in a benzo[a]pyrene-mineralizing bacterial consortium

Abstract

A bacterial consortium which rapidly mineralizes benzo[a]pyrene when it is grown on a high-boiling-point diesel fuel distillate (HBD) was recovered from soil and maintained for approximately 3 years. Previous studies have shown that mobilization of benzo[a]pyrene into the supernatant liquid precedes mineralization of this compound (R. Kanaly, R. Bartha, K. Watanabe, and S. Harayama, Appl. Environ. Microbiol. 66:4205-4211, 2000). In the present study, we found that sterilized supernatant liquid filtrate (SSLF) obtained from the growing consortium stimulated mineralization of benzo[a]pyrene when it was readministered to a consortium inoculum without HBD. Following this observation, eight bacterial strains were isolated from the consortium, and SSLF of each of them was assayed for the ability to stimulate benzo[a]pyrene mineralization by the original consortium. The SSLF obtained from one strain, designated BPC1, most vigorously stimulated benzo[a]pyrene mineralization by the original consortium; its effect was more than twofold greater than the effect of the SSLF obtained from the original consortium. A 16S rRNA gene sequence analysis and biochemical tests identified strain BPC1 as a member of the genus Rhodanobacter, whose type strain, Rhodanobacter lindaniclasticus RP5557, which was isolated for its ability to grow on the pesticide lindane, is not extant. Strain BPC1 could not grow on lindane, benzo[a]pyrene, simple hydrocarbons, and HBD in pure culture. In contrast, a competitive PCR assay indicated that strain BPC1 grew in the consortium fed only HBD and benzo[a]pyrene. This growth of BPC1 was concomitant with growth of the total bacterial consortium and preceded the initiation of benzo[a]pyrene mineralization. These results suggest that strain BPC1 has a specialized niche in the benzo[a]pyrene-mineralizing consortium; namely, it grows on metabolites produced by fellow members and contributes to benzo[a]pyrene mineralization by increasing the bioavailability of this compound.

FIG. 1.

Mineralization of 10 mg of [7-¹⁴C]benzo[a]pyrene liter⁻¹ by a bacterial consortium treated with different supernatant liquid fractions. The treatments consisted of sterile raw supernatant liquid from the parent consortium (▪), sterile supernatant liquid filtrate from the parent consortium (•), sterile supernatant liquid filter residue from the parent consortium (▴), and inoculum only without treatment (negative control) (▾). Each point represents the average for triplicate flasks, and the error bars indicate the standard deviations; error bars smaller than the symbols are not shown.

FIG. 2.

[7-¹⁴C]benzo[a]pyrene recovered from flask surfaces after incubation with strains BPC1 through BPC8. The error bars indicate the ranges for duplicates.

FIG. 3.

Mineralization of 10 mg of [7-¹⁴C]benzo[a]pyrene liter⁻¹ by a bacterial consortium treated with sterile supernatant liquid filtrates obtained from individual bacterial strains. The supernatants tested were obtained from cultures of strain BPC1 (▪), BPC2 (▿), BPC3 (▴), BPC4 (•), BPC5 (▵), BPC6 (♦), BPC7 (○), and BPC8 (□). For comparison, sterile supernatant liquid filtrates obtained from the original consortium (⋄) and from an uninoculated control (▾) were also assayed. Each point represents the average for duplicate treatments. The error bars indicate the ranges and are shown only for strain BPC1 supernatant liquid filtrate and the uninoculated control supernatant liquid filtrate for clarity.

FIG. 4.

Mineralization of 10 mg of [7-¹⁴C]benzo[a]pyrene liter⁻¹ by the bacterial consortium treated with HBD (•), supernatant liquid filtrate from strain BPC1 (▴), supernatant liquid filtrate from the original consortium (▾), and supernatant liquid filtrate from an uninoculated control (abiotic preculture) (▪). Each point represents the average for duplicate treatments, and the error bars indicate the ranges.

FIG. 5.

(A) Growth of strain BPC1 (▪) compared to the total growth of bacterial populations (•) in the benzo[a]pyrene-mineralizing consortium as determined by cPCR. (B) Time course of benzo[a]pyrene mineralization (¹⁴CO₂ evolution). The points represent the averages for triplicate determinations, and the error bars indicate the standard deviations; error bars smaller than the symbols are not shown.