Fungal Biodegradation of Endocrine Disrupting Compounds

Endocrine-disrupting compounds (EDCs) are a group of environmental pollutants known for their negative influence, particularly on aquatic organisms. EDCs tend to accumulate in aquatic organisms and also get adsorbed by sediments and on particles in the aquatic environment. These compounds mimic or antagonize the effects of endogenous hormones and hence, alter the synthesis and metabolism of natural hormones, or modify hormone receptor levels, disrupting endocrine and reproductive functions which ultimately affect the health of humans and wildlife.

Many of these chemicals have been released into the environment during the past a few decades. For example, endocrine disruption by pesticides caused an increase in salmon smolt mortality, gonad malformations in American alligators, and a decline in the seal population in the Baltic Sea (McKinlay et al. 2008). Typical EDCs of anthropogenic origin with estrogen-like action include 4-nonylphenols (NPs), bisphenol A (BPA), and 17a-ethinylestradiol (EE2) (Fig. 11.2). NPs mainly occur in the environment as degradation products of nonylphenol-polyethoxylates, which are used widely as non-ionic surfactants in many industrial applications. BPA is a raw material of some polycarbonates and epoxy resins and it is widely used as food packaging material. The compound leaches also from dental materials and it was also found in waste landfill leachates. Synthetic estrogens, such as EE2, are used as oral contraceptives. Non-metabolized EE2 and its conjugates are excreted into wastewater. During sewage treatment, EE2 is released from the corresponding conjugates by hydrolysis and reaches the environment.

Some of the latest works have focused on EDCs degradation by ligninolytic fungi as reviewed by Asgher et al. (2008). An association of compound degradation with fungal ligninolytic enzyme system was suggested in these works. However, ligninolytic fungi were found a highly efficient in removal of EDCs in aqueous media and soils using both lignin-modifying enzymes and non lignin-modifying-enzyme systems (Cabana et al. 2007b). In addition to degradation, most of the works also emphasized on changes in endocrine-disrupting activity of chemicals during the degradation. For that, sensitive and easy-to-perform in vitro bioassays were developed during the last one decade (Svobodova and Cajthaml 2010).

Purified laccase from Trametes villosa efficiently degraded BPA under in vitro conditions without requirement of mediators for the electron transfer (Fukuda et al. 2001). Structural analysis of the BPA reaction products indicated that the oligomers of BPA were formed as the result of successive oxidation-condensation (Fukuda et al. 2004). The presence of oligomers fragments, each with phenol molecules, suggested the occurrence of cleavage of the formed oligomers to release 4-isopropenylphenol. Both the soluble and insoluble fractions of the BPA reaction products had than no estrogenic activity even at high concentrations (Fukuda et al. 2004). MnP, laccase, and the laccase-mediator systems of other

bisphenol A

bisphenol A

chOh

OH HO

chOh

17a-ethinylestradiol

17a-ethinylestradiol

a- nonylphenol Fig. 11.2 Structures of anthropogenic EDCs h3C(H2C)7H2C

ligninolytic fungi are also effective in removing the estrogenic activities of BPA, NP, 17 b-estradiol (E2), and EE2 with production of high molecular weight oligomeric metabolites through radical polymerization mechanism and formation of C-C and C-O bonds (Tsutsumi et al. 2001; Suzuki et al. 2003; Lee et al. 2005). Removal of NP and BPA is associated with the production of laccase by T. versicolor and Bjerkandera sp. BOL13 (Soares et al. 2005, 2006). The enhanced biocatalytic elimination of NP, BPA and triclosan by C. polyzona by the addition of ABTS (Cabana et al. 2007a) also suggested the involvement of laccase/mediator system.

The biodegradation efficiency and estrogenic activity reduction of dibu-thylphthalate were characterized in the white rot fungi P. chrysosporium, T. versicolor and Daldinia concentrica (Lee et al. 2004). Fungi showed resistance towards the chemical and an induction of laccase in T. versicolor was observed during the dibuthylphthalate degradation. In the study of Cajthaml et al. (2009b), 4-n-nonylphenol, technical 4-nonylphenol, BPA, EE2, and triclosan were biode-graded by liquid cultures of eight ligninolytic fungal strains (I. lacteus 617/93, B. adusta 606/93, P. chrysosporium ME 446, Phanerochaete magnoliae CCBAS 134/I, P. ostreatus 3004 CCBAS 278, T. versicolor 167/93, Pycnoporus cinnabarinus CCBAS 595, and Dichomitus squalens CCBAS 750). The results enabled comparison of EDCs degradation by various fungal strains and also showed that under the used conditions, the fungi were able to degrade the EDCs within 14 days of cultivation with exception of B. adusta and P. chrysosporium in the case of triclosane and BPA, respectively. I. lacteus and P. ostreatus were found to be most efficient EDC degraders with their degradation efficiency exceeding 90 or 80%, respectively, in 7 days. Both fungi degraded technical 4-nonylphenol, BPA, and EE2 below the detection limit within first 3 days of incubation. In general, estrogenic activities assayed with a recombinant yeast test decreased with advanced degradation (Cajthaml et al. 2009b). However, in case of I. lacteus, P. ostreatus, and P. chrysosporium, the yeast assay showed a residual estrogenic activity (28-85% of initial) in EE2 cultures. Estrogenic activity in B. adusta cultures temporally increased during degradation of technical 4-nonylphenol, suggesting a production of endocrine-active intermediates. Attention was paid also to the effects of EDCs on the ligninolytic enzyme activities of the different fungal strains to evaluate their possible stimulation or suppression of activities during the biodegradation processes (Cajthaml et al. 2009b).

Liquid medium experiments with Cephalosporium aphidicola and Cunninghamella elegans were focused on identifying microbial transformation metabolites of oral contraceptives, EE2 and norethisterone (Choudhary et al. 2004). Transformation of norethisterone by C. aphidicola led to an oxidized metabolite, EE2, in this work, while the transformation of EE2 by C. elegans yielded several metabolites, 19-nor-17a-pregna-1,3,5 (10)-trien-20-yne-3,4,17b-triol, 19-nor-17a-pregna-1,3,5 (10)-trien-20-yne-3,7a,17b-triol, 19-nor-17a-pregna-1,3,5 (10)-trien-20-yne-3,11 a,17^-triol, 19-nor-17a-pregna-1,3,5 (10)-trien-20-yne-3,6b,17b-triol and 19-nor-17a-pregna-1,3,5 (10)-trien-20-yne-3,17b-diol-6b-methoxy. These metabolites were structurally characterized on the basis of spectroscopic techniques.

To scale up the degradation process, the laccase from the fungus C. polyzona was immobilized through the formation of cross-linked enzyme aggregates using polyethylene glycol and glutaraldehyde as a cross-linking agent (Cabana et al. 2007c). Enzyme preparations were then tested for their capacity to eliminate NP, BPA, and triclosan in a fluidized bed reactor which could remove all three EDCs from 5 mg/l solution. Another type of bioreactor operating with T. versicolor pellets was then used for E2 and EE2 continuous degradation (Blanquez and Guieysse 2008). In this study, E2 and EE2 were completely removed from solutions at volumetric rates of 0.16 and 0.09 mg l-1 h-1.

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