268 Polycyclic aromatic hydrocarbon bioremediation design Shigeaki Harayama Many polycyclic aromatic hydrocarbons (PAHs) are known to be mutagenic or carcinogenic, and their contamination in soil and aquifer is of great environmental concern. Limited numbers of microorganisms including mycobacteria, Sphingomonas and white rot fungi were found to be capable of degrading PAHs with four or more fused aromatic rings. In white rot fungi, lignin peroxidases are believed to be involved in the degradation of PAHs. In addition to these enzymes, P450 monooxygenases in some fungi were implicated in the degradation of PAHs. The stimulation of PAH biodegradation by the addition of surfactants was observed with some of these microorganisms although the agents were inhibitory on biodegradation with some other microorganisms. Mathematical models were constructed to explain the effect of surfactants on biodegradation. Further studies should be carried out to select the best microorganisms and surfactants for applications to PAH bioremediation. The successful application of microorganisms to the bioremediation of PAH-contaminated sites thus requires a deeper understanding of how microbial biodegradation proceeds in PAHs. In this review, the bacteria involved and the metabolic pathways for the degradation of PAHs are summarized and the biological constraints on the PAH bioremediation are discussed. PAH-degrading bacteria The biodegradation of PAHs has been observed under both aerobic and anaerobic conditions. The anaerobic biodegradation of PAHs is a slow process, and its bio- chemical mechanism has not yet been elucidated [4,5]. In contrast, aerobic biodegradative pathways, especially those for simple PAHs such as naphthalene and phenanthrene, have been extensively studied over the past decade. These pathways initiate the biodegradation of PAHs by introducing both atoms of molecular oxygen into the aromatic nucleus, the reaction being catalyzed by a mul- ticomponent dioxygenase which consists of a reductase, a ferredoxin and an iron-sulfur protein [6]. Further reactions lead to the formation of precursors of tricarboxylic acid cycle intermediates. The genes for the initial steps Addresses Marine Biotechnology Institute, 3-75-l Heita, Kamaishi, lwate 026, Japan; e-mail: harayama@kamaishi.mbio.co.jp Current Opinion in Biotechnology 1997, 8:268-273 http://biomednet.comlelecrefl0958166900600268 in the degradation of phenanthrene, naphthalene and 0 Current Biology Ltd ISSN 0958-l 669 Abbreviations LiP lignin peroxidase MnP manganese peroxidase PAH polycyclic aromatic hydrocarbon RT-PCR reverse transcriptase-PCR Introduction PAHs, which are compounds composed of two or more fused aromatic rings (Fig. l), are widely distributed in the natural environment. Coal and petroleum are two major natural sources of PAHs. Contamination by PAHs at a high level is thus found at coal and petroleum treat- ment sites, including creosote wood treatment facilities (creosote is used as a lumber preservative and contains many PAHs). PAHs, particularly the higher molecular weight types, cause great environmental concern because of their mutagenic and carcinogenic properties [1,2]. Despite this, cleaning up PAH-contaminated sites using biological treatment has not been frequently applied [3]. There are two reasons for this relative lack of bioremediation of PAH-contaminated sites: first, although biological methods have been successfully used to treat municipal and industrial waste water, their application in land remediation is still in a stage of infancy: second, PAHs are refractory to biodegradation and persist in the natural environment because of their hydrophobic nature, resulting in low water solubility and a tendency to be adsorbed to the matrix of soil and sediment. dibenzothiophene have been cloned and sequenced in many strains. The amino acid sequences of the catabolic enzymes deduced from their nucleotide sequences are 90% identical to each other. Less homologous groups of genes coding for enzymes involved in the degradation of PAHs have been found in Comamonas testosteroni [7] and zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGF Nocardioides sp. [8]. The pathway for the degradation of naphthalene generally exhibits broad substrate speci- ficity; for example, Burkholderia cepacia F297 grows on a wide variety of polycyclic aromatic compounds including fluorene, (methyl)naphthalene, phenanthrene, anchracene and dibenzothiophene. An analysis of the intermediates formed from these growth substrates has indicated that these compounds are degraded by catalytic reactions very similar to those for naphthalene degradation [9]. A new fluorene catabolic pathway has recently been found in which hydroxylation at C-9 of fluorene gen- erates 9-fluorenol, which is then dehydrogenated to 9-fluorenone [lo]. This intermediate then undergoes dioxygenation at an angular site to form l,lO-dihydro-l,lO- dihydroxyfluorene-9-one (DDF), the five-membered ring of which is subsequently cleaved to generate a substituted biphenyl. The ring-cleavage enzyme was a purified and characterized NAD+-dependent DDF dehydrogenase (NAD, nicotinamide adenine dinucleotide). A particular pathway for the degradation of dibenzothio- phene has also been described [Ill. This pathway converts dibenzothiophene to 2-hydroxybiphenyl via dibenzothio-