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Contrasting characteristics of anthracene and pyrene degradation by wood rot fungus Pycnoporus sanguineus H1

https://doi.org/10.1016/j.ibiod.2015.09.012Get rights and content

Highlights

  • The Pycnoporus sanguineus H1 strain could degrade both anthracene and pyrene.

  • Anthracene is metabolized by cytochrome P450, mycelium-associated and extracellular laccase.

  • Pyrene was degraded by cytochrome P450.

Abstract

This investigation evaluates the contrasting metabolic characteristics of anthracene and pyrene by wood rot fungus Pycnoporus sanguineus H1. Under in vivo conditions, P. sanguineus H1 degraded 67.5% of the anthracene and showed maximal laccase activity of 1568.2 U L−1. When piperonyl butoxide (PB) was added to the liquid cultures, the anthracene degradation rate increased to 73.1% and maximal laccase activity increased to 2034.3 U L−1. For pyrene, the degradation rate was 31.1%; however, after PB addition, the value decreased to 5.3% while maximal laccase activity increased to 1625.3 U L−1 from 1469.1 U L−1. Under in vitro conditions, the extracellular liquid culture (10 kDa membrane, ultra-filtered) transformed 59.9% of the anthracene, while addition of 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) enhanced the anthracene transformation rate up to 92.0%. The extracellular liquid did not convert the pyrene, however, suggesting that anthracene is modified extracellularly, (likely via laccase) while pyrene is not. Whole mycelium, as well as homogenized mycelium and microsomal proteins, transformed both anthracene and pyrene; conversion was inhibited to some extent by the PB, implying that cytochrome P450 participates in intracellular PAHs transformation. Additional evidence that ABTS enhances anthracene transformation by homogenized mycelium suggests that mycelium-associated laccase cooperates in the intracellular degradation of anthracene. These results altogether elucidate the metabolism of PAHs by P. sanguineus via extracellular laccase, intracellular cytochrome P450, and mycelium-associated laccase.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds with two or more fused benzene rings which have been identified as serious, persistent organic pollutants. The US Environmental Protection Agency (USEPA) has listed 16 PAHs as priority pollutants (Wu et al., 2010), including anthracene and pyrene. High molecular weight PAHs (HMW PAHs), such as benz[a]anthracene (BAA, four rings), benzo[a]pyrene (five rings), and dibenz[a,h]anthracene (five rings) feature the same basic structure of anthracene while exhibiting greater recalcitrant characteristics in the environment, greater toxicity, and more severe carcinogenic effects to humans than low molecular weight PAHs (LMW PAHs) (Giraud et al., 2001). Pyrene, a typical four-ring HMW PAH, exhibits teratogenicity and carcinogenicity producing mutagenicity (Wen et al., 2011). For these reasons, biodegradation studies on PAHs typically consider anthracene and pyrene to be model compounds.

Both bacteria and fungi are able to degrade PAHs (Bamforth and Singleton, 2005, Machín-Ramírez et al., 2010). Among these microorganisms, wood rot fungi are considered the most effective PAH degraders (Chupungars et al., 2009, Asgher et al., 2008), thus attracting increased attention from researchers and developers (Pointing, 2001). The most notable advantage of wood rot fungi is their secretion of extracellular ligninolytic enzymes that can extracellularly degrade substrates. Generally, these enzymes oxidize PAHs via a non-specific, radical-based reaction with corresponding quinones (Bamforth and Singleton, 2005). To date, three enzymes: lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase, have been reported to successfully catalyze PAH oxidation (Bezalel et al., 1996, Baldrian et al., 2000, Singh and Pakshirajan, 2010). Prior research conducted in our laboratory confirmed that extracellular ligninolytic enzymes of the wood rot fungus Pycnoporus sanguineus, which contains mostly laccase, can convert anthracene to anthraquinone effectively (Li et al., 2014).

In recent years, studies have suggested that cytochrome P450 monooxygenase from wood rot fungi might be involved in the initial oxidation of PAHs (Chigu et al., 2010, Ning et al., 2010). Cytochrome P450 monooxygenases, a superfamily of monooxygenases (Chigu et al., 2010), are commonly present in living organisms. The detoxification of xenobiotics is an example of several P450-dependent reactions accomplishing a series of secondary metabolic processes. This information implies that PAHs can potentially be transformed by wood rot fungi through simultaneous intracellular and extracellular pathways, which makes their metabolic process very interesting.

In this study, anthracene and pyrene were employed as PAH models. Both in vivo and in vitro conversion experiments were performed to evaluate PAH degradation potential according to different fractions of P. sanguineus. The primary goals of the experiment were to characterize the involvement of particular enzymes in PAH degradation, including ligninolytic enzymes and intracellular fraction enzymes, and to explore the possible involvement of cytochrome P-450 and mycelium-associated laccase.

Section snippets

Microorganisms, chemicals, and media

Fungus, P. sanguineus H1, was supplied by Nanjing Forestry University, China. Anthracene, pyrene, piperonyl butoxide, and 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were purchased from Sigma (St. Louis, MO, USA). All solvents used were of HPLC grade. All other chemicals and reagents used were of analytical reagent grade or higher purity.

The medium used (pH4.5) included the following: glucose 5.0 g L−1, bran 5.0 g L−1, KH2PO4 0.2 g L−1, MgSO4 0.05 g L−1, (NH4)2SO4 0.32 g L−1, CaCl2

In vivo PAH degradation by fungus

The ability of P. sanguineus H1 to degrade PAHs was evaluated in liquid culture, both with and without PB. During incubation, ligninolytic enzyme activities were assayed, but no obvious LiP or MnP activities were detected. Results indicated that the anthracene degradation capacity of this strain is quite considerable, where significant anthracene degradation occurred between 4 and 10 incubation days (Fig. 1). After 14 d of incubation, 67.5% of the total anthracene was removed. In this case, the

Acknowledgments

The work was financial supported by grants from the National Natural Science Foundation of China (41401350), Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences (SEPR2014-05) and Key Scientific Research Projects of Colleges and Universities of Henan Province.

References (25)

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