Biodegradation of Aromatic Hydrocarbons
1. Introduction
The aromatic hydrocarbons are important components of petroleum and its refined products. Aromatic compounds are broadly defined to be benzene and other compounds that exhibit similar chemical behavior (Morrison and Boyd, 1973). Benzene and substituted benzenes constitute the naturally occurring aromatic hydrocarbons. Among the most important aromatic petroleum hydrocarbons are benzene, toluene (methylbenzene), ethylbenzene, xylenes (dimethylbenzenes), and the polycyclic aromatic hydrocarbons, of which naphthalene is the simplest representative.
Since all aromatic hydrocarbons are derivatives of benzene, its properties are important. Benzene has the elemental composition C6H6 and is a flat, six-membered ring with three carbon-carbon double bonds. Because it is cyclic and unsaturated, benzene is structurally similar to the cyclic alkenes. It is, however, unusually stable and does not readily participate in reactions that are characteristic of alkenes (Morrison and Boyd, 1973). This stability results from several characteristics of benzene, including the complete delocalization of its p-bonding electrons and the way in which electrons fill its molecular orbitals, none of which will be discussed here. For a discussion of the electronic structure of benzene, see any introductory textbook on organic chemistry (e.g., Morrsion and Boyd, 1973). The unusual stability of benzene and its derivatives and the resulting effect on the reactivity of these molecules distinguishes aromatic hydrocarbons from unsaturated aliphatic hydrocarbons.
The differences between aliphatic and aromatic hydrocarbons are real and provide a useful method for categorizing these compounds. With respect to biodegradability, however, similarities also occur. For example, although anaerobic biodegradation of aromatic hydrocarbons has been reported (Grbic-Galic and Vogel, 1987; Zeyer et al., 1986), it is uncommon and slow relative to aerobic biodegradation. As is the case with aliphatic hydrocarbons, aerobic biodegradation of aromatic hydrocarbons involves the participation of molecular oxygen as a direct reactant and as the terminal electron acceptor. Finally, many important aromatic hydrocarbons can support the growth of bacteria when they are present as the sole source of carbon and energy. Although aromatic hydrocarbons are not as readily biodegradable as are normal and branched alkanes, they are somewhat more easily degradable than are the alicyclic hydrocarbons (Perry, 1984; Leahy and Colwell, 1990).
2. Biodegradation of Benzene
Because all of the important aromatic hydrocarbons that occur in petroleum are derivatives of benzene, a reaction that is common to all pathways that lead to mineralization of aromatic substrates is cleavage of the benzene ring. Therefore, this discussion of biodegradation pathways for aromatic hydrocarbons begins with a description of the pathways used for mineralization of benzene itself.
Molecular oxygen serves a reactant in two steps in the pathways for benzene catabolism. In each of these reactions, both atoms from molecular oxygen become incorporated into the substrate. Enzymes that catalyze such reactions are called dioxygenases (Sheldon and Kochi, 1981). The stoichiometry of dioxygenase-catalyzed reactions can be written as:
S + O2 ------> SO2 (R 2-2)
Note that a hydrogen donor, such as NADH, is not always required as a co-substrate in dioxygenase-catalyzed reactions, whereas hydrogen-donating co-substrates are always required for the monooxygenase-catalyzed hydroxylation of alkanes.
Ring cleavage and subsequent bacterial metabolism of benzene requires that the aromatic ring be destabilized, that is, it must be made more reactive. This is accomplished by a dioxygenase-catalyzed reaction between benzene and molecular oxygen, resulting in production of benzene dihydrodiol (i.e., cis -1,2-dihydroxycyclohexa-3,5-diene) (Ribbons and Eaton, 1982; Gottschalk, 1986). Aromaticity is restored by a dehydrogenase-catalyzed conversion of benzene dihydrodiol to catechol (i.e., 1,2-dihydroxybenzene), which is the ring cleavage substrate. The reactions leading to catechol are shown in Figure 2-8.
The dioxygenases that catalyze hydroxylation of benzene rings require an electron-donating co-substrate in order to function. The dioxygenases that catalyze the ring cleavage reactions that are described below do not require a co-substrate. The hydroxylating dioxygenases are multicomponent systems that resemble the bacterial monooxygenases: a flavoprotein accepts electrons from NADH and passes them through a ferredoxin to the
Figure 2-8. Oxidation of benzene to catechol
dioxygenase. The reduced dioxygenase reacts with O2 and the aromatic substrate (Gibson and Subramanian, 1984).
Catechol is catabolized by ring cleavage, in which the aromatic ring is broken. Ring cleavage can occur by either of two pathways: the ortho-cleavage pathway, in which the aromatic ring is split between the two carbon atoms bearing hydroxyl groups, or the meta-cleavage pathway, in which the ring is broken between a hydroxylated carbon atom and an adjacent unsubstituted carbon atom (Gottschalk, 1986). Each of these ring-cleavage reactions is catalyzed by a dioxygenase. The subsequent metabolic pathways are quite different, but they both lead to TCA cycle intermediates (acetate and succinate) or to substrates that can be easily converted to TCA cycle intermediates (pyruvate and acetaldehyde). The ortho-cleavage pathway (also called the b-ketoadipate pathway) is shown in Figure 2-9, and the meta-cleavage pathway is presented in Figure 2-10.
3. Biodegradation of Alkylbenzenes
Alkyl-substituted benzenes, such as toluene, ethylbenzene, and the xylenes, are common environmental contaminants, because they are important contaminants of gasoline and are widely used as solvents and intermediates in chemical synthesis (see Section I.D.). These compounds can serve as the sole sources of carbon and energy for a variety of
Figure 2-9. Ortho- cleavage pathway for catabolism of catechol.
bacteria, including members of the Pseudomonas , Achromobacter, and Nocardia genera (Gibson and Subramanian, 1984). Metabolism of alkylbenzenes may be initiated by oxidation of either the alkyl side chain or the aromatic ring.
Growth of Pseudomonas aeruginosa on toluene is an example of a catabolic pathway that is initiated by side-chain oxidation (Ribbons and Eaton, 1982; Gibson and Subramanian, 1984). In a monooxygenase-catalyzed reaction, toluene is converted to benzyl alcohol, which is further oxidized to benzoic acid by dehydrogenation. Benzoic
Figure 2-10. Meta- cleavage pathway for catechol catabolism.
acid is the substrate for insertion of oxygen into the aromatic ring, leading to production of catechol. The reactions leading to catechol are shown in Figure 2-11. Catechol cleavage proceeds as was described above.
Enzymes that catalyze the hydroxylation of aromatic methyl groups are not well studied. In some cases, the reaction may be catalyzed by a non-specific alkane monooxygenase. The n-alkane monooxygenases of P. aeruginosa and of several Nocardia sp. are examples of this (Gibson and Subramanian, 1984). Many alkane monooxygenases, however, cannot hydroxylate methyl groups that are attached to aromatic rings. The converse is also true: a monooxygenase that hydroxylates xylene methyl groups is inactive against n-alkanes (Higgins and Gilbert, 1978).
Oxidation of toluene and ethylbenzene by Pseudomonas putida provides an example of the other pathway by which alkyl-substituted benzenes are degraded: initiation by dioxygenase-catalyzed ring hydroxylation, leading to 3- or 4-methylcatechols (or the analogous ethylcatechols) (Ribbons and Eaton, 1982; Gibson and Subramanian, 1984). The alkylcatechols are further oxidized by meta-cleavage. Similar pathways for toluene oxidation are observed in Pseudomonas mildenbergii , an Achromobacter sp., and Nocardia corallina. The reactions involved are outlined in Figure 2-12.
Alkyl substituents larger than ethyl, such as isopropyl, n-butyl, and isobutyl, hinder oxidation of the benzene ring by P. putida. Substituted benzenes such as these are slowly oxidized to cis -dihydrodiols, which are subsequently metabolized through the meta-cleavage pathway. Although oxidized slowly, these substrates support growth of bacteria that initiate degradation of alkyl-substituted benzenes by ring hydroxylation. Dialkyl-benzenes, such as m- and p-xylene, do not (Gibson and Subramanian, 1984). These are
converted to dimethylcatechols, which are not cleaved by the P. putida catechol-2,3-dioxygenase. N. corallina can catalyze the ortho-cleavage of 3,6-dimethylcatechol to 2,5-dimethyl-cis,cis -mucconic acid, but can degrade this product no further (Gibson and Subramanian, 1984).
A Nocardia sp. can grow on o-xylene by ring hydroxylation, leading to 3,4-dimethylcatechol. This organism oxidizes the dimethylcatechol by a meta-cleavage pathway, leading ultimately to acetic acid, propionaldehyde, and pyruvate (Gibson and Subramanian, 1984). This Nocardia sp. is unusual in its ability to oxidize dimethylcatechols by meta-cleavage and in its ability to grow on o-xylene. This Nocardia sp. is one of the few that has been reported to use o-xylene as the sole source of carbon and energy. In contrast, isolating bacteria that can grow on m- or p-xylene is relatively easy.
Figure 2-11. Oxidation of toluene to catechol by Psuedomonas aeruginosa.
Figure 2-12. Toluene metabolism by P. putida : Ring hydroxylation pathway.
Most bacteria that grow on m- or p-xylene first oxidize a methyl group, producing the corresponding toluic acid, and then oxidize the aromatic ring. Oxygen insertion occurs at the carbon atom bearing the carboxyl group, and decarboxylation accompanies dehydrogenation of the diol to methylcatechol (Ribbons and Eaton, 1982). The oxygen insertion/decarboxylation reactions are analogous to those used by P. aeruginosa to degrade toluene (Figure 2-11). The biodegradation pathways for m-xylene is shown in Figure 2-13.
Figure 2-13. Biodegradation of m- xylene by P. putida PaWI and Pseudomonas Pxy.