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Keywords:

  • ectomycorrhizal fungus;
  • PAH;
  • Gadgil-effect;
  • Pinus sylvestris;
  • degradation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    Polycyclic aromatic hydrocarbons (PAHs) are an important class of persistent organic pollutants (POPs) in the environment and accumulate in forest soils. These soils are often dominated by ectomycorrhizal (EcM) roots, but little is known about how EcM fungi degrade PAHs, or the overall effect of field colonized EcM roots on the fate of PAHs.
  • • 
    The ability of eight EcM fungi to degrade PAHs in liquid culture spiked with 14C labelled PAHs was investigated. Microcosms were used to determine the impact of naturally colonized mycorrhizal pine seedlings on PAH mineralization and volatilization.
  • • 
    Only two EcM fungi (Thelephora terrestris and Laccaria laccata) degraded at least one PAH and none were able to mineralize the PAHs in pure culture. Where degradation occurred, the compounds were only mono-oxygenated. EcM pine seedlings did not alter naphthalene mineralization or volatilization but retarded fluorene mineralization by 35% compared with unplanted, ectomycorrhizosphere soil inoculated, microcosms.
  • • 
    The EcM fungi possessed limited PAH degrading abilities, which may explain why EcM dominated microcosms retarded fluorene mineralization. This observation is considered in relation to the ‘Gadgil-effect’, where retarded litter decomposition has been observed in the presence of EcM roots.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chemicals

14C labelled and unlabelled PAHs and authentic standards of potential metabolites (3,4-dihydroxybenzoic acid, 2-hydroxybenzoic acid, 1,2-dihydroxynaphthalene, 2-hydroxynaphthalene, 1-hydroxynaphthalene, 1,2-benzenedicarboxylic acid, 4-hydroxy-1-indanone, 1-indanone, 9-hydroxyfluorene, 2-hydroxy-9-fluorenone, 2-hydroxyfluorene, 9-fluoreneone, 1-hydroxypyrene) were obtained from Sigma-Aldrich Co. Ltd, Gillingham, UK. HPLC grade solvents were obtained from Rathburn Chemicals Ltd, UK.

Liquid culture study

Fungal culture  Eight EcMF (Rhizopogon luteolus (Tannar), Thelephora terrestris (DGC-3(2)1), Lactarius turpis (LT001), Laccaria laccata (LL002), Cenococcum geophilum (54), Suillus variegatus (AS), and Paxillus involutus (PI014 and DGC-4(2))) obtained from existing culture collections, and isolated from unpolluted sites, were grown at 22°C in 90 mm diameter Petri dishes containing pH 5.5 modified Melin-Norkans agar medium (MMN) of the following composition (NH4)2HPO4 (0.5 g), KH2PO4 (0.3 g), MgSO4·7H2O (0.14 g), CaCl2·6H2O (0.05 g), NaCl (0.025 g), Fe EDTA (0.0125 g), glucose (10 g), thiamine (0.01 mg), No. 3 agar (Oxoid) 15 g made up to 1 l with distilled H20 and adjusted to pH 5.4 with HCl. Liquid cultures were prepared by aseptic transfer of two 5 mm diameter EcMF plugs, from actively growing colony margins, to 100 ml amber glass Wheaton bottles containing 10 mL MMN medium without agar. The bottles were covered with sterile aluminium foil and incubated at 20°C for 14 d (28 d for T. terrestris).

Growth of EcMF with PAHs  EcMF were grown with either naphthalene, fluorene or pyrene. PAH/14C-PAHs, dissolved in hexane, were added to the Wheaton bottles to give a final PAH concentration of 10 µg ml−1 and an activity of 0.5 kBq ml−1 in the culture medium. Because the specific activities of 14C stock solutions varied, the final quantities of hexane added to the bottles were 54 µl (naphthalene), 118 µl (fluorene) and 139 µl (pyrene). Nonradioactive replicates were also prepared for HPLC-MS analysis. Test tubes (50 × 10 mm) containing 1.0 ml 3M NaOH(aq) were placed into each Wheaton bottle to trap evolved 14CO2. The bottles were sealed with aluminium crimp-caps, fitted with aluminium faced silicone septa, and incubated at 20°C for 28 d (42 d for T. terrestris). Controls were prepared containing killed inoculum and no inoculum. Three replicates were prepared for each EcMF/PAH combination.

Mineralization of PAHs  A 100-µl aliquot of trap-NaOH(aq) was added to 2 ml HionicFluor scintillation fluid (Packard Bioscience Ltd, Beaconsfield, UK) and its 14C activity counted on a scintillation counter (Minaxi Tri-Carb 4000 Series, Packard Bioscience) following extraction of dissolved organic 14C by shaking with 1 ml hexane.

Identification of metabolites in culture media  The MMN medium was filtered through glass-wool to remove fungal biomass and the filtrate collected in a 10-ml SPE reservoir. Supelclean LC-18 (100 mg : 1 ml) SPE tubes (Supelco, USA) were conditioned with 1.0 ml methanol followed by 1.0 ml dd.H2O before the media filtrate was passed through the tubes from the reservoirs. no. 14C activity was detected in postextracted media. Tubes were eluted with 2 × 0.5 ml acetonitrile that had been used to wash the Wheaton bottle and septa. Analysis was performed on 100 µl injections of the extracts using an 1100 series HPLC-MSD (Agilent Technologies, Stockport, UK) with a UV diode-array detector (all UV-absorption units (AU) relate to this machine using Agilent Technologies Chemstation software). Separation of metabolites was conducted with a Hypersil® (ThermoQuest, Runcorn, UK) ODS HPLC column (250 × 4.6 mm, 5 µm) at 20°C. The solvent gradient (water = A, acetonitrile = B) was 80% A, 20% B at 0 min to 30% A, 70% B at 60 min at 2 ml min−1. After separation, the mobile phase was negatively ionized by API-ES (gas temperature = 350°C, drying gas flow rate = 12 l min−1, nebuliser pressure = 40 psig, quadrupole temperature = 100°C, capillary voltage = 3000 V) and scanned from 50 to 300 m z−1 with a fragmentor voltage of 100 vs. A library of authentic potential PAH metabolite standards was also compiled using the same conditions. PAH metabolites were detected by diverting 14C labelled samples to a Beta-RAM 14C detector (LabLogic, Sheffield, UK). To improve 14C detection, selected extracts were concentrated c. 10 × by Kuderna-Danish evaporation.

Microcosm study

Collection and preparation of material  Ectomycorrhizal Scots pine seedlings (< 1 yr old, 40–50 mm tall) were collected from a Scots pine plantation in Aberdeenshire (57 : 06 : 51 N, 2 : 56 : 06 W) and the majority of soil carefully removed from the root system. Mycorrhizosphere organic matter (hereafter referred to as ‘OM’) was also collected at the same site by shaking the F-horizon soil from seedling roots. On return to the lab, the seedlings were planted, three per pot, in a 5 : 1 : 1.5 (v : v : v) mixture of vermiculite, OM and MMN solution (containing 0.1 g l−1 glucose) and grown for 28 d. The remaining OM was sealed in a plastic bag and stored at 3°C until required. Nonmycorrhizal pine seedlings were prepared by sterilizing seeds with 100 volume H2O2 for 20 min with one drop of Decon-90 as a surfactant. The seeds were then washed thoroughly with sterile deionized water and germinated on MMN-agar plates. Plates with contaminated seeds were discarded and uncontaminated seeds were transferred to sealed autoclaved pots in a 5 : 1 : 1.5 (v : v : v) vermiculite, moss-peat, carbon-free MMN substrate and grown on until they were the same size as the field collected seedlings.

Microcosm construction Microcosms were constructed entirely from glass with Quickfit™ connections to allow progressive measurement of 14C volatilization and mineralization in a flow-through system (Fig. 1). Filter sterilized air (0.45 µm) was drawn continuously into the top of each microcosm (18 cm3 min−1 by a multichannel Watson-Marlow™ peristaltic pump), through the growth chamber, over a mineral-oil coated glass-wool volatile trap (150 g glass-wool coated with 20 ml mineral oil added in 1 L hexane. The hexane was evaporated off and a total of 7.2 g of the oil coated wool was added to each trap). The air was finally pulled through a NaOH(aq) CO2 trap (10 ml, 1 M NaOH). All microcosms were autoclaved prior to assembly.

image

Figure 1. Construction of glass, flow-through microcosms.

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Experimental design  The microcosms contained either (1) sterile OM/vermiculite with unsterile OM (unplanted) (2) sterile OM/vermiculite planted with unsterile field-inoculated pine seedlings with added unsterile OM (mycorrhizal) or (3) sterile OM/vermiculite planted with a sterile (nonmycorrhizal) pine seedling (control). The PAHs (naphthalene/naphthalene-UL-14C or fluorene/fluorene-9–14C) were spiked into 100 g autoclaved (1 h, 121°C) OM/vermiculite/MMN substrate to give a concentration of 30 µg g−1 and activity of 300 Bq g−1. To distribute the PAHs evenly throughout the substrate, they were added in 0.5 ml hexane to a 5-g subsample of the substrate, which was then mixed with the bulk soil in a sterile, glass-stoppered flask (Reid et al., 1998). 2 g fresh OM was added to the nonsterile treatments at this mixing stage. The inoculated and/or spiked substrate was then transferred directly into the bottom growth chamber compartment. Sterile or field-inoculated pine seedlings were carefully transplanted into the microcosms before final assembly as described above (Fig. 1) and incubated in a Sanyo® (MLR-350) growth cabinet (15°C, 18 h light period). For each PAH treatment, field-inoculated seedlings were taken from the same pot to reduce variation in EcM colonization. Three replicates were prepared for each treatment/PAH combination.

Sampling  Glass-wool and CO2 traps were refreshed every 7–28 d depending on the stage of the experiment and PAH. To quantify volatilized organic-14C, the glass-wool was divided into three parts (top, middle and bottom) and the activity of each part was recorded, after addition of 8 ml Ultima-Gold scintillation fluid (Packard Bioscience Ltd). For all measurements, there was negligible activity in the final (bottom) piece of glass-wool, which demonstrates that all volatile organic compounds were removed by the top and middle glass-wool sections. Mineralized 14C was measured in a 100-µl aliquot of the NaOH(aq) trap after the addition of 1 ml Hionic-Fluor scintillation fluid. Naphthalene microcosms were sampled over 12 h and fluorene microcosms for 45 h, after which, they were destructively harvested to quantify and characterize the remaining 14C activity. Substrate-bound organic 14C was Soxhlet extracted (50°C, 6 h, 400 ml 1 : 1 hexane/acetone) and quantified before 80 × condensation by Kuderna-Danish evaporation of the solvent at 60°C. Condensed extracts were then analysed for potential PAH metabolites by 14C-HPLC as described in the liquid culture method above. Shoot and root material was digested separately by Dalal digestion (Dalal, 1979) (10 ml chromic acid with a 1-mL 3M NaOH(aq)-trap autoclaved for 1 h at 121°C) to quantify bound and incorporated 14C.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Degradation of PAHs by EcMF in liquid culture

None of the EcMF mineralized the PAHs because there was no 14C recovery in the NaOH(aq) traps (data not shown), but L. laccata degraded naphthalene and fluorene and T. terrestris degraded fluorene to more polar metabolites (see below). There was no evidence of PAH degradation by the remaining EcMF isolates and no degradation products were detected in the fungus-free or killed-fungus controls (data not shown). Pyrene was not degraded by any of the isolates. Replicate chromatograms were consistent within fungal/PAH treatments.

Degradation of naphthalene by L. laccata14C-HPLC analysis of the L. laccata acetonitrile extracts resulted in three peaks with retention times (RTs) of 34.0, 20.4 and 18.4 min corresponding to naphthalene and metabolites 1 and 2, respectively (Table 1). The RTs, UV-spectra and mass spectra of metabolites ‘1’ and ‘2’ corresponded with authentic standards of 1-hydroxynaphthalene and 2-hydroxynaphthalene, respectively. The concentration of 2-hydroxynaphthalene was below the detection limits of the Beta-RAM 14C detector.

Table 1.  Characterization and identification or naphthalene, fluorene and pyrene metabolites produced by Laccaria laccata and Thelephora terrestris
A –L. laccata
Compound14CRT/minUV max/umm/z of fragment ions (% RI)Standard matchEvidencea
Naphthalene
Metabolite 2No18.4233143 (100)2-hydroxynaphthaleneRT, UV, MS
Metabolite 1Yes20.4212, 231, 295143 (100), 144 (11.0)1-hydroxynaphthaleneRT, UV, MS
Fluorene
Metabolite 5Yes19.2200, 272No ionisation9-hydroxyfluoreneRT, UV
Metabolite 4No27.0200, 271, 309181 (100), 182 (12.6)2-hydroxyfluoreneRT, UV, MS
Metabolite 3Yes30.2257No ionisation9-fluorenoneRT, UV
Pyrene
No metabolism
B –T. terrestris
Compound14CRT/minUV max/umm/z of fragment ions (% RI)Standard matchEvidence
  1. RT, HPLC retention time; UV, UV spectrum; MS, mass spectrum.

Naphthalene
No metabolism
Fluorene
Metabolite 5Yes19.2200, 272No ionisation9-hydroxyfluoreneRT, UV
Metabolite 3Yes30.2257No ionisation9-fluorenoneRT, UV
Pyrene
No metabolism

Degradation of fluorene by L. laccata and T. terrestris14C-HPLC analysis of the L. laccata acetonitrile extracts resulted in four peaks with RTs of 43.0, 30.2, 27.0 and 19.2 min corresponding to fluorene and metabolites 3–5, respectively. The RTs and UV-spectra of metabolites 3–5 corresponded with authentic standards of 9-fluorenone, 2-hydroxyfluorene and 9-hydroxyfluorne, respectively (Table 1). Metabolite 4 also had a molecular m/z of 181, which corresponds to the negative molecular ion of 2-hydroxyfluorene. Mass spectra could not be obtained for metabolites 3 and 5. T. terrestris degraded fluorene to metabolites 3 and 5, but metabolite 4 was not detected.

Degradation of PAHs in microcosms

More than 90% of the root tips of the field inoculated pine seedlings were colonized by EcMF at the final harvest of each experiment with extensive rhizomorph formation. The sterile seedlings remained nonmycorrhizal.

Mineralization and volatilization of naphthalene  Naphthalene mineralization was detected after 48 h in the nonsterile (OM inoculated) microcosms and after 96 h in the sterile pine treatment. The presence of EcM pine seedlings in the microcosms with added OM had no effect on the rate, or total mineralization (c. 25% added 14C activity), of naphthalene. There was considerably less mineralization (10% added 14C activity) in the sterile pine controls (F2,6 = 40, P < 0.001) (Fig. 2a). Only 10% of the naphthalene volatilized from the nonsterile microcosms, whereas c. 40% volatilized from the sterile pine controls (Fig. 2b). Very little 14C was recovered from the substrate by Soxhlet extraction (< 3% of the recovered 14C) (Fig. 3a) and no metabolites were detected.

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Figure 2. Mineralization (a) and volatilisaton (b) of naphthalene-14C from the unplanted (open circles, dotted line), mycorrhizal (solid circles, solid line) or sterile control (open circles, solid line) microcosms. Values expressed as percent of spiked 14C activity (Error bars, 1 SE; n= 3).

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image

Figure 3. Final proportion of 14C volatilized, mineralized and bound to substrate in microcosms spiked with (a) naphthalene-14C (12 h incubation) or (b) fluorene-14C (45 h incubation) (Error bars, 1 SE; n= 3).

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Mineralization and volatilization of fluorene Fluorene was mineralized after 15 h in the nonsterile treatments but no mineralization was detected in the sterile pine treatment after 45 h (Fig. 4a). In the nonsterile treatments, the rate of fluorene mineralization was significantly slower in the microcosms planted with EcM pine seedlings such that 35% less fluorene was mineralized over the experimental period (F1,4 = 15, P= 0.019). Volatilization of fluorene was unaffected by treatments (Fig. 4b) and there were no differences in total losses (Fig. 3b). Approximately 50% of the recovered 14C was extracted from the substrate by Soxhlet extraction. The majority of this recovery was parent fluorene, although 2-hydroxyfluorene, 9-hydroxyfluorene, 2-hydroxy-9-fluorenone and 9-fluorenone were also detected (Fig. 5). The presence of EcM pine had no effect on the metabolite profile in the unsterile substrates, although no 2-hydroxyfluorene and c. 10 × more 9-hydroxyfluorene (F2,6 = 85, P < 0.001) were extracted from the sterile pine treatment. There was also c. 10 × more 9-fluorenone in the sterile pine treatment extracts (F2,6 = 4.6, P= 0.061).

image

Figure 4. Mineralization (a) and volatilisaton (b) of fluorene-14C from the unplanted (open circles, dotted line), mycorrhizal (solid circles, solid line) or sterile control (open circles, solid line) microcosms. Values expressed as percent of spiked 14C activity (Error bars, 1 SE; n= 3).

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image

Figure 5. Fluorene and fluorene metabolites recovered from the substrate following 45 h incubation in unplanted, mycorrhizal and sterile control microcosms (Error bars, 1 SE; n= 3).

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Given the difference in fluorene mineralization between planted and unplanted nonsterile treatments, substrate gravimetric moisture content and pH were determined at the final harvest to check for differences in physical conditions between treatments. The substrate in the mycorrhizal pine microcosms was drier (5%) than in the unplanted and sterile control treatments (F2,6 = 5.02, P= 0.052) (Fig. 6). There was no difference in substrate pH (4.23 ± 0.093) between treatments (F2,6 = 0.22, P= 0.812).

image

Figure 6. Moisture content of substrate spiked with fluorene following 45 h incubation in unplanted, mycorrhizal and sterile control microcosms (Error bars, 1 SE; n= 3).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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 sylvestrisS. 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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank Francis Martin (INRA-Nancy), Jan Poskitt (CEH Merlewood), Sara Preston and Luis Villareal-Ruiz (University of Aberdeen) for supplying the EcMF cultures. This work was funded by BBSRC-BIRE award (BRE13662).

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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