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Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutant (POP) in the environment because of their potential toxicity, mutagenicity, and carcinogenicity (Wilson & Jones, 1993). Strong adsorption to soil particles and low water solubility limit the bioavailability and therefore the rate of PAH biodegradation in soil. PAHs are produced naturally by incomplete combustion of organic matter but also occur at high local concentrations as a result of human activities such as coal-gas production and accidental oil spillages. As a result of their hydrophobicity, PAHs accumulate in forest soils by adsorption to tree leaves/needles and subsequent incorporation into the litter layer (Simonich & Hites, 1994).
POP degradation by nonmycorrhizal white-rot fungi has been extensively studied (Barr & Aust, 1994; Song, 1997; Kim et al., 1998), although their use as efficient remediation agents is limited because the fungi must be inoculated along with a finite source of carbon, such as wood-chips, which are difficult to distribute throughout the soil volume. A range of basidiomycete and ascomycete fungi form symbiotic ectomycorrhizal (EcM) associations with woody plant roots (Smith & Read, 1997). Ectomycorrhizal fungi (EcMF) may overcome the problems associated with free-living white-rot fungal remediation because they are distributed throughout the soil by roots, and provided with a long-term supply of photosynthetic carbon from their hosts. In addition, the development of root systems may improve soil aeration and drainage, which increases biological activity and assists POP volatilization.
Some, but not all, EcMF are able to degrade/mineralize a range of POPs both in axenic culture (Meharg et al., 1997; Braun-Lullemann et al., 1999; Gramss et al., 1999; Green et al., 1999) and in their symbiotic state (Meharg et al., 1997). It has been suggested that, like white-rot fungi, this is caused by the production of extracellular, nonspecific, oxidative enzymes that enable degradation of complex aromatic polymers, such as lignin, in natural soils (Barr & Aust, 1994; Meharg & Cairney, 2000). However, there is limited evidence for the production of lignin peroxidase by EcMF (Cairney et al., 2003).
It may be that EcM fungi are generally less able to degrade complex polyphenolic compounds (e.g. lignin) compared with free-living wood decomposing fungi such as white-rot fungi (Bending & Read, 1997; Wu et al., 2003). There are also reports of retarded litter decomposition rates in the presence of EcM roots; a phenomenon know as the ‘Gadgil’ effect (Bending, 2003). This effect has been attributed to a number of mechanisms, including competitive displacement of free-living saprotrophic microbes (Lindahl et al., 2001) and soil drying by ectomycorrhizal roots and mycelium (Koide & Wu, 2003). It is therefore unclear whether natural EcM communities have a positive or negative effect on POP degradation in the natural environment.
The aim of this study was to investigate the potential of EcMs to remediate PAH contaminated soils. Since little is known about the degradation pathways of POPs by EcMF (Green et al., 1999), we first used 14C-labelled PAHs, in combination with 14C-HPLC and LC-MS, to determine how eight diverse EcMF degrade PAHs in liquid culture. We then used specially constructed microcosms to study the fate of 14C-labelled PAHs in a substrate inoculated with F-horizon forest-soil, with Scots pine (Pinus sylvestris L.) seedlings with and without a naturally established complement of EcMF.
PAH degradation by mycorrhizas is often inferred by measuring loss of parent compound relative to control treatments. However, biological activity and substrate characteristics can have a profound effect on POP partitioning between adsorbed, dissolved and volatile fractions and hence confound interpretations based solely on parent compound recovery. The construction of our microcosms, and use of 14C-labelled PAHs, allowed progressive quantification of PAH volatilization and mineralization, followed by characterization of the remaining transformed and untransformed parent compounds.
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This study has demonstrated that some EcMF have a limited ability to degrade PAHs in pure culture and that pine seedlings with natural EcM communities have a negative impact on fluorene mineralization in a microcosm.
A range of EcMF are known to degrade POPs (Meharg & Cairney, 2000), although until now, metabolic pathways have only been determined for 4-fluorobiphenyl (Green et al., 1999). In the present study, two of the eight EcMF isolates, L. laccata and T. terrestris, were able to degrade one or more PAH and the metabolites they produced have been characterized. L. laccata degraded naphthalene to 1- and 2-hydroxynaphthalene. Microbial degradation of naphthalene normally occurs through dioxygenation prior to PAH ring-cleavage (Smith, 1994; Stringfellow & Aitken, 1995). Both L. laccata and T. terrestris metabolized fluorene to 9-fluorenone and 9-hydroxyfluorene. These metabolites are also produced when the white-rot fungus Pleurotus ostreatus degrades fluorene (Bezalel et al., 1996a) and are considered to be ‘dead-end’ products formed by the action of cytochrome P-450 monooxygenase (Bezalel et al., 1996b). Accumulation of these metabolites is not uncommon and has also been demonstrated for a Mycobacterium sp. and Pseudomonas cepacia F297 (Boldrin et al., 1993; Grifoll et al., 1995). In addition to these methylenic group metabolites, there was evidence that L. laccata produced 2-hydroxyfluorene (metabolite 2). Although nonmethylenic group monohydroxyfluorenes have not been detected in other microbial degradation studies, the bacteria Pseudomonas sp. F274 and Arthrobacter sp. F101 cleave the aromatic rings of fluorene following 1,2- or 3,4-dihydroxylation (Grifoll et al., 1994; Casellas et al., 1997). It appears that the monohydroxylated metabolites produced by the EcMF do not lead to ring-cleavage and accumulate as further dead-end products. This suggests, at least from the isolates screened here, that EcMF do not have the enzymatic capabilities to dihydroxylate fluorene. It is noteworthy that dihydroxylation of fluorene aromatic rings by Arthrobacter sp. F101 also failed to produce subsequent oxidation products once the C-9 carbonyl group had been oxidized (Casellas et al., 1997). None of our isolates were able to degrade pyrene, although removal of pyrene from liquid media has been reported elsewhere (Braun-Lüllemann et al., 1997; Gramss et al., 1999). The lack of degradation reported in the present study may either reflect differences in culture conditions or variation in degradation capabilities between EcMF strains. Alternatively, these previous reports of pyrene degradation may be methodological artefacts, caused by loss of pyrene via a route other than degradation (e.g. adsorption to fungal hyphae).
Metabolism without mineralization confirms previous reports that pure cultures of EcMF degrade POPs to dead-end metabolites, which may themselves present problems of accumulation and toxicity (Green et al., 1999). However, although EcMF may not be able to cleave aromatic-rings, it has been suggested that partial POP degradation products may be substrates for further metabolism by other soil organisms (Sarand et al., 1999). Indeed, ring hydroxylation is the thermodynamically limiting step to ring-cleavage by bacteria (Meharg & Cairney, 2000). For example, combinations of white-rot fungi with soil-microorganisms result in greater mineralization of pyrene than either groups (der Wiesche et al., 1996) and degradation of m-toluate has also been observed in Pinus sylvestris–S. bovinus rhizospheres inoculated with a toluene degrading Pseudomonas fluorescens (Sarand et al., 1999).
The microcosm study determined the effect of mycorrhizal pine on PAH degradation in systems which contained mycorrhizal fungi along with associated mycorrhizosphere competent bacteria and nonmycorrhizal fungi. There were substantial losses of naphthalene and fluorene through both mineralization and volatilization from the microcosms and a marked difference in these rates of loss between the two PAHs. Naphthalene volatilized, and was mineralized, more rapidly than fluorene, to the extent that very little naphthalene remained in the substrate at the end of the study. This was not surprising and largely reflects the different physico-chemical characteristics of the two PAHs.
Mineralization of fluorene was substantially suppressed by mycorrhizal pine seedlings compared with the unplanted microcosms. This was not caused by enhanced volatilization in the planted microcosms. To be sure that this effect was caused by the EcM fungi and not to the presence of the seedling per se would require a nonmycorrhizal control with added unsterile OM. This was not possible to achieve, but there is no evidence in the literature that plant roots suppress POP degradation and no likely mechanism comes to mind. However, rates of litter decomposition can be reduced in the presence of EcM roots (Gadgil & Gadgil, 1971; Gadgil & Gadgil, 1974). Current explanations are reviewed by Bending (2003), but in brief, have been attributed to direct competition between saprotrophs and EcMF, either as a result of resource competition (e.g. Suillus variegatus and Paxillus involutus are able to scavenge phosphorus from the wood-decomposing fungus, Hypholoma fasciculare (Lindahl et al., 1999)) or direct inhibition through production of antimicrobial compounds (Marx, 1973). Alternatively, Koide & Wu (2003) suggest that EcM roots retard litter decomposition by reducing substrate moisture content. Although we measured a small reduction in soil moisture content in the microcosms containing mycorrhizal pine seedlings, it is unlikely that such a small difference resulted in the observed mineralization suppression of our study. Given the limited ability of the EcMF screened in liquid culture to degrade PAHs and their limited ability to degrade complex polyphenolic compounds compared with free-living saprotrophic fungi (Bending & Read, 1997), it is possible that dominance of the planted microcosms by EcMF at the expense of free-living saprotrophs caused the retarded fluorene mineralization. It is less likely that a build up of dead-end metabolites, as happens in liquid culture, retarded mineralization, since there was no difference in the metabolites extracted from the planted and unplanted substrates. Although we found suppression of PAH degradation, other authors have found that EcM pine roots may enhance the density of bacteria and expedite the removal of POPs from ectomycorrhizosphere soil (Heinonsalo et al., 2000). In other studies, it has been observed that bacterial mineralization of PAHs is considerably reduced, or even completely stopped, by the presence of other, more easily metabolized, organic substrates in the medium (Juhasz et al., 2000). This effect was attributed to a change in the bacterial metabolism to accommodate the more easily catabolized organic material at the expense of the more energy demanding PAH catabolism. It may be that carbon provided to the microcosm by seedling exudation alters microbial (free-living and mycorrhizal) metabolism such that PAH catabolism is considerably reduced, or shut down completely.
In contrast to fluorene, there were no differences in naphthalene volatilization or mineralization between the mycorrhizal and unplanted microcosms at any time throughout the study. The greater rate of volatilization in the sterile control microcosms is probably caused by greater quantities of available naphthalene as a result of the reduced mineralization. The rapid volatilization of naphthalene (compared with fluorene) from the microcosms may, in part, have been caused by a combination of low organic content of the substrate (resulting in less substrate bound naphthalene) and the constant flow of air through the substrate. This may have resulted in loss of naphthalene from the microcosms before a mycorrhizosphere capable of enhancing, or retarding, naphthalene mineralization developed. Interestingly, in a similar microcosm study conducted in this laboratory, 100% forest soil OM was used, and naphthalene volatilization was considerably reduced (< 3.5%). Consequently, mineralization was reduced from 45% in the unplanted treatments to 20% in the field colonized mycorrhizal pine treatments (P < 0.001) (L.A. Uffindell, pers. comm.).
We did not identify the EcMF that colonized the planted microcosms, or characterize the ability of these EcMF to degrade PAHs. However, we have demonstrated that EcMF from a number of genera have little or no ability to degrade PAHs in pure culture and similar patterns might be expected in natural EcM populations. We are currently conducting further microcosm studies to determine whether similar retarded fluorene mineralization is observed when pine seedlings are preinoculated with the fluorene degrading T. terrestris isolate identified in the liquid culture study. The influence of EcMs on POP degradation may also be determined by the availability of soil carbon for saprotrophic growth. Many industrially polluted soils have very low levels of carbon substrates to support free-living saprotroph growth (Barr & Aust, 1994). In such soils, even slow rates of POP degradation by EcMs may be beneficial as a long-term remediation strategy compared with unplanted soils.
This study has demonstrated that the ability of EcMF to degrade PAHs is not ubiquitous but restricted to certain species or isolates. None of the metabolites were mineralisable, probably because of the inability of the fungi to produce dioxygenated metabolites. Under the microcosm conditions employed in this study, fluorene mineralization was retarded by EcM pine seedlings, mirroring the ‘Gadgil-effect’ observed in many litter decomposition studies. EcMs therefore may not be efficient PAH remediation agents, although further studies are required to determine the effects of soil characteristics on remediation efficiency. The microbial communities that developed in the ectomycorrhizospheres in our microcosms are likely to differ from those of an undisturbed soil. It is possible that interactions between EcMF and their undisturbed microbial communities may result in different outcomes to those observed in our study. Finally, it must be noted that within the high diversity of EcMF, some, particularly those isolated from polluted sites, may have greater PAH degrading capabilities than the isolates examined in this study.