Biodegradation of Polycyclic Aromatic Hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous compounds, with two or more fused benzene rings arranged either in a linear or a cluster mode, derived from both natural and anthropogenic sources. Several members of this class of compounds have been included among priority pollutants owing to their toxic, mutagenic and carcinogenic properties (Haritash and Kaushik 2009).

The persistence of these compounds in the environment is mainly due to their low solubility in water and stable polycondensed aromatic structure. Hydrophobicity and recalcitrance of PAHs to microbial degradation generally increase as the molecular weight increases. Besides being toxic to animals, some PAHs with four or more benzene rings, such as benzo[a]anthracene, chrysene and benzo[a]pyrene, have been shown to be carcinogenic (Bezalel et al. 1996a). In the last few decades, different approaches to PAHs biodegradation have been investigated and white rot basidiomycetes have attracted significant interest since the pioneering studies with Phanerochaete chrysosporium (Hammel et al. 1986). Later on, several other fungal species have been demonstrated to metabolize PAHs significantly under model liquid culture conditions or in the soil and the most studied and efficient strains of ligninolytic fungi are P. chrysosporium, Trametes versicolor, Pleurotus ostreatus, Bjerkandera adusta, Irpex lacteus and Panus tigrinus (Pointing 2001; Novotny et al. 2009; Baldrian 2008; Covino et al. 2010). It was proved that these fungi can oxidize PAHs under in vitro conditions using Lac and ligninolytic peroxidases (Majcherczyk et al. 1998; Eibes et al. 2006; Baborova et al. 2006). In addition, the use of either natural or synthetic mediators was described (Sack et al. 1997; Johannes and Majcherczyk 2000; CaƱas and Camarero 2010; Camarero et al. 2008). Besides, the extracellular degradation pathway, cytochrome P-450 monooxygenase and epoxide hydrolase in the initial degradation have been proved to be active in the degradation (Bezalel et al. 1997). Using 14C-labeled compounds, it was proved that ligninolytic fungi are able to mineralize PAHs completely to carbon dioxide (Bezalel et al. 1996b; Wolter et al. 1997). However, intermediates arisen from ring-cleavage reactions that were triggered by fungi are hardly described. Previously, Hammel et al. (1991) showed that P. chrysosporium was able to decompose anthracene into phthalic acid that was identified as ring-fission product. Bezalel et al. (1996c) presented the mechanism of 2,2'-diphenic acid production from phenanthrene. These authors suggested that cytochrom P-450 of P. ostreatus was responsible for the attack on phenanthrene enabling further ring opening reactions. Moen and Hammel (1994) reported formation of 2,2'-diphenic acid from phenan-threne after lipid peroxidation by MnP. Majcherczyk and co-workers found several ring-cleavage products of acenaphthylene and acenaphthene after incubation with Lac as well as a laccase-mediator system of T. versicolor (Johannes et al. 1998; Majcherczyk et al. 1998). In a later study (Cajthaml et al. 2002), the degradation of phenanthrene, anthracene, fluoranthene and pyrene by liquid culture of I. lacteus (degradation extent after 50 days: 67-95%) provided several metabolites that pointed to ring-cleavage processes during degradation. Structures of some of the compounds suggested involvement of both enzymatic systems, P-450 and ligninolytic peroxidases (Fig. 11.1).

Later, in vitro experiments were carried out using MnP from the same fungus and proved that this enzyme is able to degrade the representatives of PAHs (Baborova et al. 2006). Major degradation products of anthracene were identified and the results showed a new role of MnP in PAH degradation by I. lacteus when a ring fission product was detected 2-(2'-Hydroxybenzoyl)-benzoic acid. A pathway similar to anthracene has been also published for benzo(a)anthracene resulting

COOH

COOCH3

Fig. 11.1 Pathway proposed for anthracene degradation by I. lacteus with indication of possible enzyme involvement

COOH

COOCH3

Fig. 11.1 Pathway proposed for anthracene degradation by I. lacteus with indication of possible enzyme involvement

(Table 11.1) in phthalic acid via several intermediates possessing two aromatic rings e.g., 1,4-naphthalenedione, 1,4-naphthalenediol and 1,2,3,4-tetrahydro-1-hydroxynaphthalene (Cajthaml et al. 2006).

Similar findings were documented also in case of P. tigrinus when the best results were obtained in shaking cultures where PAHs were degraded by 91 and 97% in complex maltextract-glucose media and nitrogen limited mineral media, respectively, within 4 weeks. In stationary cultures, on the contrary, the degradation was never higher than 50%. This fungus was also able to decompose aromatic structures and a set of various products after ring-cleavages were detected (Covino et al. 2010). The presence of some degradation products (e.g., hydrox-ylated derivatives of anthrone and phenanthrene 9,10-dihydrodiol) suggested the possible involvement of cytochrome P-450 monooxygenous and epoxide hydrolase system, the active form of which was found in 7-day-old cultures on the complex rich media. In vitro experiments showed that the MnP from P. tigrinus had a wider PAH substrate range and higher oxidation ability than the laccase produced by the same strain.

Table 11.1 Structural suggestion and mass spectral characteristics of BaA metabolites

Structural suggestion

MW (CI)

m/z of fragment ions (relative intensity)

Benz(a)anthracene-7,12-

258

258 (100), 230 (41), 202 (47.2), 174 (4.5), 150 (4.6)

dionea

1-tetralonea

146

146 (75.9), 131 (13), 118 (100), 104 (3.4), 90 (26.9),

77 (8.7)

1,2,3,4-tetrahydro-1-

148

148 (8.4), 147 (20.1), 130 (100), 119 (42.4), 105 (20.2),

hydroxynaphthalenea

91 (25), 77 (5.9)

4-hydroxy-1-tetralone

162

162 (18.6), 145 (17), 134 (68.2), 115 (15.8), 105 (100),

77 (24.1), 51 (15.8)

4-hydroxy-1-tetralone-TMS

234

219 (100), 189 (8.8), 115 (10.2)

Phthalic anhydrideab

148

148 (2.3), 104 (100), 76 (41.2), 50 (20.4)

Phthalic acid di-TMSa

310

310 (3.7), 295 (57.6), 265 (6.4), 221 (27.5), 193 (3.8),

147 (100), 73 (53.1)

Monomethyl phthalic acida

163

163 (15.4), 149 (60.7),136 (14.2), 104 (100), 92 (19.5),

76 (96.7)

Monomethyl phthalic acid-

252

252 (2.2), 237 (100),163 (50),133 (7.5), 89 (77.7)

TMSa

Phthalidea

134

134 (12.7), 105 (100), 77 (40.9), 51 (9.0)

Dimethyl phthalic acida

194

194 (3.0), 163 (100), 133 (15.8), 77 (9.7)

Naphthalene-1,4-dionea

158

158 (100), 130 (38.9), 104 (62.9), 102 (60.5), 76 (45.1)

1,4-dihydroxynaphthalenea

160

160 (85.4), 131 (21.5), 104 (100), 76 (41.5)

1,2-naphthalic anhydrideb

198

198 (80.1), 154 (87.9), 126 (100)

a Structures were later identified with authentic standard b Dehydrated form of the metabolite a Structures were later identified with authentic standard b Dehydrated form of the metabolite

P. ostreatus, well known as oyster mushroom, has been many times described as efficient degrader of PAHs, as well (Bezalel et al. 1996b; Wolter et al. 1997). Grown in the presence of several PAHs and their analogues (benzo[a]pyrene, pyrene, fluorene, phenanthrene, anthracene), it was able to metabolize and in some cases to mineralize them. Among the metabolites identified were: phenanthrene trans-9,10-dihydrodiol and 2,2'-diphenic acid from phenanthrene, pyrene trans-4,5-dihydrodiol from pyrene and anthracene trans-1,2-dihydrodiol and 9,10-anthraquinone from anthracene. For instance, the fungus was shown to be able to decompose phenanthrene, anthracene and pyrene by 50, 92 and 35%, respectively, in bran flakes media in 5 days (Pickard et al. 1999). Schutzendubel et al. (1999) found that during 3 days of incubation, B. adusta removed 56 and 38% of fluorene and anthracene, while P. ostreatus degraded 43 and 60% of these compounds; other PAHs were degraded to a lower extent. Except for anthracene in cultures of P. ostreatus, all PAHs were removed uniformly during the cultivation time but fluorene and anthracene were degraded faster than other PAHs in basidiomycete's rich media. The detected intermediates were mostly keto compounds.

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