Degradation of aroclor 1242 dechlorination products in sediments by Burkholderia xenovorans LB400(ohb) and Rhodococcus sp. strain RHA1(fcb)

Abstract

Burkholderia xenovorans strain LB400, which possesses the biphenyl pathway, was engineered to contain the oxygenolytic ortho dehalogenation (ohb) operon, allowing it to grow on 2-chlorobenzoate and to completely mineralize 2-chlorobiphenyl. A two-stage anaerobic/aerobic biotreatment process for Aroclor 1242-contaminated sediment was simulated, and the degradation activities and genetic stabilities of LB400(ohb) and the previously constructed strain RHA1(fcb), capable of growth on 4-chlorobenzoate, were monitored during the aerobic phase. The population dynamics of both strains were also followed by selective plating and real-time PCR, with comparable results; populations of both recombinants increased in the contaminated sediment. Inoculation at different cell densities (10(4) or 10(6) cells g(-1) sediment) did not affect the extent of polychlorinated biphenyl (PCB) biodegradation. After 30 days, PCB removal rates for high and low inoculation densities were 57% and 54%, respectively, during the aerobic phase.

FIG. 1.

Degradation of defined artificial PCB mixtures M (A) and C (B) by recombinant B. xenovorans strain LB400(ohb) (S1) and its wild type (S2). Bars shown for S3 correspond to noninoculated controls. Data are mean values from duplicate samples.

FIG. 2.

Aroclor 1242 congener distribution and concentration (μg g⁻¹) in contaminated sediment at time zero (A) or after 1 year of incubation under anaerobic conditions (B), followed by aerobic incubation for 30 days with recombinant strains RHA1(fcb) and LB400(ohb) at 10⁶ cells g⁻¹ of sediment (C) or 10⁴ cells g⁻¹ (D). See reference for PCB congeners represented by each peak.

FIG. 3.

Population densities of recombinant strains Rhodococcus sp. strain RHA1(fcb) (•) and B. xenovorans strain LB400(ohb) (▾), containing the fcb and ohb operons, respectively, inoculated into Aroclor 1242-contaminated sediment that had previously undergone anaerobic PCB dechlorination. Cell numbers were also estimated by real-time PCR with TaqMan fcb (□) and TaqMan ohb (▵) probes. (A) Inoculation density of 10⁶ cells g⁻¹ sediment for each strain. (B) Inoculation density of 10⁴ cells g⁻¹ sediment for each strain. (C) Noncontaminated control with inoculation density of 10⁴ cells g⁻¹ sediment for each strain. (D) Noninoculated control. The value above each sampling time represents the stability of the fcb or ohb operon in 10 randomly chosen colonies from each strain when isolation was possible above the detection limit. Where error bars are not shown, the standard error of triplicates was smaller than the size of the symbol.

FIG. 4.

Percentages of PCBs remaining during aerobic treatment with recombinant strains RHA1(fcb) and LB400(ohb) after incubation with two different inoculum densities. Symbols: •, 10⁶ cells of each strain g⁻¹ of sediment; ▾, 10⁴ cells of each strain g⁻¹ of sediment; ▪, noninoculated control. Duplicate samples were used for the noninoculated control. Where error bars are not shown, the standard error of triplicates was smaller than the size of the symbol.