Degradation Products of Polychlorinated Biphenyls and Their In Vitro Transformation by Ligninolytic Fungi

Abstract

Metabolites of polychlorinated biphenyls (PCBs)-hydroxylated PCBs (OH-PCBs), chlorobenzyl alcohols (CB-OHs), and chlorobenzaldehydes (CB-CHOs)-were incubated in vitro with the extracellular liquid of Pleurotus ostreatus, which contains mainly laccase and low manganese-dependent peroxidase (MnP) activity. The enzymes were able to decrease the amount of most of the tested OH-PCBs by > 80% within 1 h; the removal of more recalcitrant OH-PCBs was greatly enhanced by the addition of the laccase mediator syringaldehyde. Conversely, glutathione substantially hindered the reaction, suggesting that it acted as a laccase inhibitor. Hydroxylated dibenzofuran and chlorobenzoic acid were identified as transformation products of OH-PCBs. The extracellular enzymes also oxidized the CB-OHs to the corresponding CB-CHOs on the order of hours to days; however, the mediated and nonmediated setups exhibited only slight differences, and the participating enzymes could not be determined. When CB-CHOs were used as the substrates, only partial transformation was observed. In an additional experiment, the extracellular liquid of Irpex lacteus, which contains predominantly MnP, was able to efficiently transform CB-CHOs with the aid of glutathione; mono- and di-chloroacetophenones were detected as transformation products. These results demonstrate that extracellular enzymes of ligninolytic fungi can act on a wide range of PCB metabolites, emphasizing their potential for bioremediation.

Keywords: Irpex lacteus; Pleurotus ostreatus; biodegradation; chlorobenzaldehydes; chlorobenzyl alcohols; hydroxylated PCBs.

Figure 1

Residual amounts (related to corresponding heat-deactivated controls) of hydroxylated polychlorinated biphenyls (OH-PCBs) obtained during the in vitro experiment with the extracellular liquid of Pleurotus ostreatus: (a) 4-hydroxy-2-chlorobiphenyl and 4-hydroxy-3-chlorobiphenyl; (b) 2-hydroxy-5-chlorobiphenyl; (c) 4-hydroxy-4’-chlorobiphenyl; (d) 2-hydroxy-3,5-dichlorobiphenyl; (e) 3-hydroxy-2’,5’-dichlorobiphenyl; (f) 4-hydroxy-2’,5’-dichlorobiphenyl; (g) 4-hydroxy-3,5-dichlorobiphenyl; (h) 4-hydroxy-2,2’,5’-trichlorobiphenyl; and (i) 4-hydroxy-2’,4’,6’-trichlorobiphenyl. The OH-PCBs were incubated in a mixture; the initial concentration was 2 µg mL⁻¹ for each. The initial enzyme activities were 450 U L⁻¹ of laccase and 30 U L⁻¹ of manganese-dependent peroxidase (MnP). The laccase-favouring setup (Lac) contained no mediator (◼), syringaldehyde (SA; ✕), or 1-hydroxybenzotriazole (HBT; ◯); the MnP-favouring setup contained no mediator (▲) or glutathione (GSH; ⯁).

Figure 2

Residual amounts (related to corresponding heat-deactivated controls) of chlorobenzyl alcohols (CB-OHs) obtained during the in vitro experiment with the extracellular liquid of Pleurotus ostreatus: (a) 2-chlorobenzyl alcohol; (b) 3-chlorobenzyl alcohol; (c) 4-chlorobenzyl alcohol; (d) 2,3-dichlorobenzyl alcohol; (e) 2,4-dichlorobenzyl alcohol; (f) 2,5-dichlorobenzyl alcohol; (g) 2,6-dichlorobenzyl alcohol; (h) 3,4-dichlorobenzyl alcohol; (i) 3,5-dichlorobenzyl alcohol; and (j) 2,4,6-trichlorobenzyl alcohol. The CB-OHs were incubated in a mixture; the initial concentration was 2 µg mL⁻¹ for each. The initial enzyme activities were 450 U L⁻¹ of laccase and 30 U L⁻¹ of manganese-dependent peroxidase (MnP). The laccase-favouring setup (Lac) contained no mediator (◼), syringaldehyde (SA; ✕), or 1-hydroxybenzotriazole (HBT; ◯); the MnP-favouring setup contained no mediator (▲) or glutathione (GSH; ⯁).

Figure 3

Residual amounts (related to corresponding heat-deactivated controls) of chlorobenzaldehydes (CB-CHOs) detected after incubation with the extracellular liquid of Irpex lacteus with no mediator (no med) or reduced glutathione (GSH): 2-chlorobenzaldehyde (2-CB-CHO); 3-chlorobenzaldehyde (3-CB-CHO); 4-chlorobenzaldehyde (4-CB-CHO); 2,3-dichlorobenzaldehyde (2,3-CB-CHO); 2,4-dichlorobenzaldehyde (2,4-CB-CHO); 2,5-dichlorobenzaldehyde (2,5-CB-CHO); 2,6-dichlorobenzaldehyde (2,6-CB-CHO); 3,4-dichlorobenzaldehyde (3,4-CB-CHO); 3,5-dichlorobenzaldehyde (3,5-CB-CHO); and 2,3,6-trichlorobenzaldehyde (2,3,6-CB-CHO). The CB-CHOs were incubated in a mixture; the initial concentration was 2 µg mL⁻¹ for each. The initial enzyme activities were 706 U L⁻¹ of manganese-dependent peroxidase and 114 U L⁻¹ of manganese-independent peroxidase. The asterisks denote where significant (p-value < 0.05) differences were found between 1 h and 24 h or between 1 h and 72 h (ANOVA and Tukey’s range test).

Figure 4

Pathways of the biotransformation of PCBs and their metabolites by ligninolytic fungi as proposed in the in vivo and in vitro studies by Čvančarová et al., Kamei et al., Muzikář et al., Schultz et al., Fujihiro et al., Kordon et al., Stella et al., and Ferreira et al. [1,2,4,18,19,20,21,31] and in this work. Where possible, enzyme involvement is stated. The asterisks denote compounds under study in the present work and the detected metabolites. MnP = manganese-dependent peroxidase; AAO = aryl-alcohol oxidase.