Erwin Grund a, Annegret Scbmitz b, J~rg Fiedler b and Karl-Heinz Gartemann b
" G B F - Gesellschaft fiir Biotechnologische Forschung mRH, Mascheroder Weg 1, D-38124 Braunschweig, Germany bUniversit~t Bielefeld, Fakult~it fiir Biologie,
Gentechnologie/Mikrobiologie, Postfach 100131, Universit~tsstraBe, D-33594 Bielefeld 1, Germany
I N T R O D U C T I O N
Polycyclic aromatic hydrocarbons (PAHs) occur as natural constituents and combustion products of fossil fuels and, therefore, are ubiquitous environmental contaminants [1-3]. Furthermore, coal-tar creosote, a chemical which mainly consists of PAHs, has been widely used as a wood preservative for over 150 years, and accidental spillage or improper disposal of creosote has resulted in extensive contamination of soil, surface waters, and groundwater aquifers [4]. PAHs may have toxic, mutagenic, and carcinogenic properties [5,6] and, therefore, the fate of these x e n o b i o t i c s in nature is of environmental concern.
Polyhalogenated dibenzo-p-dioxins and the closely related dibenzofurans are unintentionally produced as contaminating by-products during the manufacture of pesticides, incineration of halogen-containing aromatic chemicals and bleaching of paper pulp [7-9]. Halogen-containing aromatic compounds are common constituents of industrial and domestic waste, thus polyhalogenated dibenzo-p-dioxins and dibenzofurans are formed in incineration plants. These highly toxic and mutagenic compounds [10,11] have, therefore, become widespread contaminants of the environment. Another interesting point, with respect to contamination of the environment with chlorinated dioxins, is that these compounds may be enzymatically formed from chlorinated phenols [12] by the action of peroxidases.
The third example for polycyclic aromatic compounds of environmental concern is the polychlorinated biphenyls (PCBs). The vast majority of PCBs in the environment are derived from commercial mixtures (Aroclors),
which contain 60 to 80 different congeners. These m~xtures have found widespread industrial use in the past, owing to their physical and chemical stability and their dielectric properties. Inadequate waste disposal has led to their release into the environment, and they have been routinely detected in soil and water samples since the early 1960s.
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Their toxicity has caused PCBs to be prohibited in many countries.
These compounds are some of the most serious environmental pollutants, and numerous studies have focused on their toxicity, mutagenicity, bioaccumulation, environmental fate, and health risks [13-15].
Many of the above mentioned different types of polycyclic aromatic compounds are recalcitrant, and bioaccumulation occurs. However, recent work has shown that these chemicals can be degraded oxidatively by some bacterial isolates.
Complete mineralization of chlorinated polycyclic aromatic compounds, like dibenzo-p-dioxins, dibenzofurans, and biphenyls, requires the presence of two sets of genes, one set for the degradation of the polycyclic aromatic structure and a second set for the degradation of chlorinated monocyclic aromatics. Previous work has shown that more efficient degradation of 4-chlorobiphenyl occurs in a co-culture, which contains a bacterial strain that is able to degrade 4-chlorobenzoate produced by a 4-chlorobiphenyl-degrading strain [16,17]. Therefore, we will present data on the aerobic dehalogenation of chlorobenzoates by bacteria.
During the degradation of chlorinated dibenzofurans by bacterial strains, chlorinated salicylates will be accumulated, and during the degradation of chlorinated dibenzo-p-dioxins, chlorinated phenols will be accumulated.
The degradation of these halogenated aromatics has been discussed in another chapter of this book.
N A P H T H A L E N E
It is well known that microorganisms can degrade naphthalene and other PAHs. Reports exist, for example, on the aerobic degradation of the tricyclic PAHs, phenanthrene and anthracene [18-20], and the tetracyclic PAHs, pyrene [21,22] and fluoranthrene [23,24] by Gram- negative and Gram-positive soil bacteria. Even the aerobic attack of fungi on PAHs has been reported [25-27]. These few examples show that PAHs can be degraded aerobically, and that the biological breakdown of these human health-risk compounds may represent a promising approach for the detoxification of PAH-con~m~nated areas. But, as was shown recently, parameters, like adsorption of PAHs by soil particles [28], may influence the biodegradability of PAHs. This shows that one of the problems, that arises during the breakdown of PAHs by microorganisms, is the availability of these hydrocarbons for the microorganisms. The mechanisms, by which microorganisms make available the PAHs, are not fully understood todate. Therefore, we will focus on the enzymatic reactions, that occur during the aerobic breakdown of PAHs. The principle, by which the different PAHs are degraded aerobically by bacteria, is always quite similar. Because of this similarity of the degradation routes for different PAHs, it is possible to understand the principles
105 while explaining one of the best understood degradation routes of PAHs which is the oxidative catabolism of naphthalene.
It is well established t h a t bacteria can oxidatively metabolize naphthalene and, therefore, the degradation route of naphthalene and even the genetics of this naphthalene catabolism are well understood.
The different degradation routes for naphthalene are presented in Figure 1. In 1964, the metabolic sequence of enzymatic reactions, leading to the degradation of naphthalene, was first presented by Davies and Evans [29]. Later studies have shown that "cis-naphthalene
dihydrodiol" is the first metabolite in the bacterial metabolism of naphthalene [30]. The enzyme, that catalyzes this reaction, is the naphthalene dioxygenase [31]. The aerobic catabolism of homocyclic aromatic compounds, that do not contain hydroxy groups, is usually initiated by the action of a dioxygenase. In general, dioxygenases are enzymes that activate molecular oxygen and introduce two hydroxy- groups. All bacterial dioxygenases, that catalyze the formation of dihydrodiols and that have been analyzed in detail, consist of two or three iron-containing enzyme components, and require NADH or NADPH as electron donors. The naphthalene dioxygenase consists of three protein components, an iron-sulphur flavoprotein, a two-iron, two- sulphur ferredoxin, and an iron-sulphur protein, which are essential for cis-naphthalene dihydrodiol formation. The naphthalene dioxygenase also accepts indole as a substrate which leads to the formation of indigo [32]. The second step in the bacterial oxidation of naphthalene is the conversion of cis-naphthalene dihydrodiol to 1,2-dihydroxynaphthalene.
This reaction is catalyzed by cis-naphthalene dihydrodiol dehydrogenase and requires NAD as an electron acceptor [33].
1,2-Dihydroxynaphthalene is enzymatically cleaved by an oxygenase [34], and by the action of an isomerase and an aldolase, salicylaldehyde and pyruvate are formed [29,35,36]. Salicylaldehyde is oxidized to salicylate by the action of a NAD-dependent dehydrogenase [37].
The reactions, described so far, are derived from the most extensively studied catabolic pathway for naphthalene, which is encoded by the NAH7 plasmid of Pseudomonas putida. But, the same sequence of reactions seems to take place in all naphthalene-degrading bacteria, which means that salicylate is a common intermediate in naphthalene catabolism. With respect to the enzymes involved in the breakdown of naphthalene, there seem to exist differences between the bacterial species, that can grow on naphthalene-containing media. A recently isolated and described Rhodococcus sp., for example, seems to have a 1,2-dihydroxynaphthalene oxygenase that requires NADH [38].
All known bacterial species, that can grow on naphthalene as sole source of carbon and energy, can degrade salicylate as well. Three different catabolic routes are known for the total degradation of salicylate (Figure 1), and all these reaction sequences are realized in naphthalene-
106
H OH OH
I II III
C~iO H F 0~-~ ~'CHO * HOOc ''~'kO = OH
CH 3
VIi VI
OH
viii