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Published in J. Environ. Qual. 33:45-53 (2004).
© ASA, CSSA, SSSA
677 S. Segoe Rd., Madison, WI 53711 USA

TECHNICAL REPORTS

Bioremediation and Biodegradation

Pyrene Degradation in Forest Humus Microcosms with or without Pine and its Mycorrhizal Fungus

Teija T. Koivulaa,d, Mirja Salkinoja-Salonenb, Rainer Peltolaa,b and Martin Romantschuk*,c

a Department of Biosciences, Division of General Microbiology, P.O. Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland
b Department of Applied Chemistry and Microbiology, P.O. Box 56 (Viikinkaari 9), FIN-00014 University of Helsinki, Finland
c Department of Ecological and Environmental Sciences, University of Helsinki, Niemenkatu 73, FIN-15140 Lahti, Finland
d VTT Biotechnology, Tietotie 2, Espoo, P.O. Box 1500, FIN-02044 VTT, Finland

* Corresponding author (romantsc{at}mappi.helsinki.fi).

Received for publication April 4, 2002.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mineralization potential of forest humus and the self-cleaning potential of a boreal coniferous forest environment for polycyclic aromatic hydrocarbon (PAH) compounds was studied using a model ecosystem of acid forest humus (pH = 3.6) and pyrene as the model compound. The matrix was natural humus or humus mixed with oil-polluted soil in the presence and absence of Scots pine (Pinus sylvestris L.) and its mycorrhizal fungus (Paxillus involutus). The rates of pyrene mineralization in the microcosms with humus implants (without pine) were initially insignificant but increased from Day 64 onward to 47 µg kg–1 d–1 and further to 144 µg kg–1 d–1 after Day 105. In the pine-planted humus microcosms the rate of mineralization also increased, reaching 28 µg kg–1 d–1 after Day 105. The 14CO2 emission was already considerable in nonplanted microcosms containing oily soil at Day 21 and the pyrene mineralization continued throughout the study. The pyrene was converted to CO2 at rates of 0.07 and 0.6 µg kg–1 d–1 in the oily-soil implanted microcosms with and without pine, respectively. When the probable assimilation of 14CO2 by the pine and ground vegetation was taken into account the most efficient microcosm mineralized 20% of the 91.2 mg kg–1 pyrene in 180 d. The presence of pine and its mycorrhizal fungus had no statistically significant effect on mineralization yields. The rates of pyrene mineralization observed in this study for forest humus exceeded the total annual deposition rate of PAHs in southern Finland. This indicates that accumulation in forest soil is not to be expected.

Abbreviations: PAH, polycyclic aromatic hydrocarbon


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
POLYCYCLIC AROMATIC HYDROCARBONS are ubiquitous pollutants not only in urban and industrialized areas but also in remote places (Edwards, 1983; Kawamura and Suzuki, 1994). Accumulation has been shown in rural areas through atmospheric deposition (Thomas et al., 1984; Wild and Jones, 1995). The environmental PAH load mostly results from combustion of fuels (Howsam and Jones, 1998). Soils and sediments are the major repositories for PAHs. The fate of PAHs in soils varies from volatilization to adsorption on surfaces and degradation via biotic or abiotic reactions (Cerniglia, 1992; Reilley et al., 1996). Microbial attack may convert PAHs into more toxic or mutagenic compounds (Lambert et al., 1995; Wild and Jones, 1995) than the parent PAH thus presenting a risk for human health and ecosystems.

Microbial degradation of many PAH compounds has been shown, but the PAHs with the lowest solubility in water tend to resist degradation in the environment (Cerniglia, 1993; Kanaly and Harayama, 2000). Many PAH mineralization studies have been conducted in soils and sediments (Carmichael and Pfaender, 1997; Grosser et al., 1995; Hambrick et al., 1980; Heitkamp and Cerniglia, 1987; Herbes, 1981; Herbes and Schwall, 1978; Klinge et al., 2001). Few degradation studies have been performed in acidic forest soils (Guthrie and Pfaender, 1998; Roper and Pfaender, 2001; Wild and Jones, 1993). Boreal conifer forest soils are acidic and highly humic. They cover the major part of cold northern areas where the atmospheric PAHs deposit due to low-temperature condensation. It is important to know if and how PAHs can be mineralized in such soils.

We analyzed mineralization of pyrene in microcosms of natural humus and oil-polluted soil in the presence and in the absence of Scots pine and its mycorrhizal fungus Paxillus involutus. Scots pine was chosen because it is one of the most common tree species in conifer forests. Most if not all of the conifer trees are living in symbiosis with mycorrhizal fungi. Conifers have been found to be highly dependent on mycorrhizal fungi for plant nutrient uptake (Smith and Read, 1997). Few studies have been published concerning the use of mycorrhizal fungi in symbiosis with a plant for the degradation of organic pollutants (Dittmann et al., 2002; Joner et al., 2001; Meharg et al., 1997). We studied pyrene mineralization in forest humus and the self-cleaning potential of the conifer forest environment for PAH compounds.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Growth Substrates and the Spiking of Humus
Humus (Ao horizon) was collected from a Scots pine forest in southern Finland. The pine forest had no known previous exposure to PAH other than by distant transport. The humus contained 320 g kg–1 (dry wt.) of total organic C and 12 g kg–1 (dry wt.) of total N, and the pH was 3.6 (in 0.1 M KCl). Oily soil was collected from an industrial oil waste site in southern Finland. The outdoor industrial land farming site had been used for waste oil from a refinery for more than 20 yr. This soil contained 39 g of petroleum hydrocarbons, 1.5 mg of pyrene and methylated pyrenes, 81 g of total organic C, and 3.4 g of total N kg–1 (dry wt.), and its pH was 6.0 (in 0.1 M KCl). The soils were sieved (3.3-mm sieve), mixed thoroughly, and stored at +4°C in the dark until used.

Preparation of Two-Dimensional Microcosms
Scots pine seeds (Seed Lot R01-72-1685; Foundation for Forest Tree Breeding, Läyliäinen, Finland) were propagated into sterile seedlings and infected with the ectomycorrhizal fungus P. involutus (Hintikka, 1988) as described by Timonen et al. (1993). Ten-week-old mycorrhizal seedlings were transferred onto a humus layer of 5 mm pressed onto the back plate of an acrylic (400 cm2) two-dimensional microcosm (Fig. 1a ; Finlay and Read, 1986; Timonen et al., 1997). The humus layer was protected from light. Similar microcosms were also prepared omitting the pines (Fig. 1b). The microcosms were allowed to propagate in a growth room with day and night temperatures of +20 and +13°C, respectively, and a 20-h photoperiod with a photon flux of 220 µmol m–2 s–1. Microcosms were watered regularly to keep the humus moist.



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  		Fig. 1. (a) Two-dimensional microcosm (without the front acrylic plate; the thickness of the humus layer is 5 mm) photographed about two months after planting the Scots pine seedling (4.5 months old) infected with fungus Paxillus involutus. (b) Nonplanted microcosm. Note the mosses growing at the edges of the microcosms and mycorrhizas (marked with arrowheads). When the microcosms were about one month old, an area of 25 cm2 (marked with asterisks) was excised (Region I) and replaced by an implant consisting of natural humus with pyrene (0 or 100 mg kg–1) or a mixture of natural humus and waste oil soil as described in Table 1. The implanted microcosms were equilibrated in the growth room for one month. Then each implant was spiked with 0.2 g of humus containing 105 disintegrations per minute (dpm) of 14C-pyrene (0.15 µg).

 

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  		Table 1. Two-dimensional microcosm setups.

 
After five weeks of mycorrhizal plant growth, a 25-cm2 region of humus (Region I, see Fig. 1) was excised and replaced with a humus implant containing 0 or 100 mg pyrene kg–1 or with a oily soil–humus mixture (which contained pyrene and methylated pyrene, 0.97 mg kg–1) as described in Table 1. The microcosms were left to propagate in a growth room as above for one month.

When the P. involutus had colonized the implanted region (Fig. 1a), 0.2 g of 14C-pyrene spiked humus (preparation of the spiked humus is explained in Table 1) was carefully added to the same region trying not to disturb the fungal growth. The microcosms without pine and fungus were treated similarly. Each microcosm was then transferred into tightly sealed glass growth chambers (Fig. 2) . Air drawn (700 mL h–1) from the microcosms with a peristaltic pump (Model 502 Peristaltic Pump Type 110; Watson-Marlow Ltd., Falmouth, England) was bubbled through 100 mL of 2 M NaOH to trap the CO2 and through an activated carbon (2 g) trap to collect the exhaust volatile organic compounds (Fig. 2). The NaOH solution was changed once in three weeks.



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  		Fig. 2. Design of the growth chamber for collecting 14CO2 and volatile organic compounds. Each glass chamber (260 x 40 x 330 mm) contained one microcosm. The chambers were connected to wash bottles of glass containing 2 M NaOH to trap CO2 and an activated carbon trap. Air was withdrawn from the growth chamber and pumped through the traps at 700 mL h–1.

 
Reference microcosms with pines were set up with L-[U- 14C]-glutamic acid as described in Table 1. Microcosms with and without a mycorrhizal plant and no 14C label were prepared to monitor the background radioactivity (see Table 1).

Abiotic Degradation of Pyrene
Humus (20 g dry wt.) containing pyrene (100 mg kg–1) and 14C-pyrene (1.7 kBq, 0.15 µg) was placed in 1-L glass bottles. Three replicates were prepared with sterile humus and three with unsterile humus. The emitted CO2 was trapped into NaOH solution as described above. The flasks were incubated in the dark at +23°C for 170 d.

Sampling of the Two-Dimensional Microcosms
The 25-cm2 implants of spiked humus (Region I) and the rest (Region II) of the humus were sampled separately (Fig. 1). The needles, stem, and roots of the pines were separated. The roots in Region I were separated from those grown in Region II to avoid 14C contamination from the soil. All roots were washed with distilled water. The needles, stems and roots of the pines were sliced, ground in liquid nitrogen, and stored at –20°C until used. Ground vegetation (mostly mosses) growing at the edges of the microcosms was collected and dried.

Radioactivity Measurement
The contents of the CO2 trap (Fig. 2) were acidified with concentrated H2SO4. The gaseous CO2 formed was driven by air bubbling into two bottles connected in series, each holding 8 mL of Lumasorb II (Lumac-LSC BV, Groningen, the Netherlands). Scintillation fluid (8 mL, Carboluma; Lumac-LSC BV) was added and the radioactivity measured using a Wallac 1415 liquid scintillation counter (Wallac Oy, Turku, Finland).

The 14CO2 produced in the flasks holding sterile humus was measured by adding 1 mL of the NaOH in the trap into 19 mL of scintillation solution (Opti Phase HiSafe 3; Wallac Oy). Radioactivity was measured as above.

The 14C in the pulverized plant material, dried (60°C) oily soil, and humus was measured by burning an aliquot in an oxidizer (Junitek Oy, Turku, Finland). The produced CO2 was trapped into Lumasorb II solution, scintillation fluid (Carboluma) was added, and the radioactivity measured. The burning efficiency was 92% measured by burning known standards of 14C-palmitate or 14C-cholesterol (Wallac Oy).

Solvent Extraction
Pulverized needles (0.30–0.35 g) or roots (0.21–0.23 g) were suspended in 5 mL of chloroform and methanol (2:1 v/v) (Rathburn Chemicals Ltd., Walkerburn, Scotland) and shaken at room temperature overnight (Folch et al., 1957). The extracts were centrifuged at 3000 x g for 10 min, the supernatants harvested, and the bottom phase reextracted. Five milliliters of deionized water was added to the combined supernatants and the phases were allowed to separate. The 14C activity of the upper aqueous phase was measured with Instagel II plus scintillation fluid (Packard Bioscience BV, Groningen, the Netherlands). Chloroform was evaporated, the residue burned in a Junitek oxidizer, and the 14CO2 measured as described above. The 14C in the nonextractable residue was measured after burning in the Junitek oxidizer as described above. Needles and roots from the microcosms spiked with L-14C-glutamic acid were extracted and analyzed similarly. To test the analysis method the needles were collected from nonlabeled microcosms to which 14C-pyrene was added in vitro. Recovery of the label was 86%.

Humus and soil were extracted similarly to the plant material. The recovery (86%) of the method was determined by spiking the humus with a known amount of 14C-pyrene.

Statistical Analysis
The natural logarithm of the CO2 efflux data and of the total amount of pyrene metabolized data (see Table 2) was calculated to reduce the variance. These transformed data were used in all statistical analyses, except in the calculation of the Z values (see below). The CO2 efflux data and the total amount of pyrene metabolized data in the pine series (PH/91.2, PO/0.95, and PH/0.07 microcosms) and in the nonpine series (H/91.2, O/0.95, and H/0.07 microcosms) were analyzed separately by one-way analysis of variance (ANOVA). As ANOVA revealed highly significant differences in both of the measured outcomes in pine and nonpine series, the t test was used to analyze the differences of the three experiments within the pine and nonpine series.


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  		Table 2. Distribution of 14C in the different compartments of two-dimensional microcosms 180 d after spiking with 14C-pyrene.

 
Two-way ANOVA was used to analyze the effect of pine vs. nonpine and the three categories of implants on the total amount of pyrene metabolized, and a marginally significant interaction (P = 0.068; 2 df) between the two variables was found. Because of this and the similarity in the results, the two highest pyrene concentration groups of the pine series (PH/91.2 and PO/0.95) were combined and compared with those of the nonpine series (H/91.2 and O/0.95) by two-way ANOVA adjusting for the implant type. The effect of pines on the implants containing the lowest amount of pyrene (PH/0.07 vs. H/0.07) was analyzed by t test. However, even after the logarithmic transformation, the variances of PH/0.07 and H/0.07 differed significantly from each other (P = 0.04 for equal variances). Therefore, the analysis was continued as follows. The nonpine results for H/0.07 were considerably more accurate (Table 2) corresponding the normal distribution with mean ± SD of 0.179 ± 0.021%. Using this nonpine distribution as a basis for comparison, the Z values of the three individual replicates of the PH/0.07 microcosm were calculated to evaluate whether they would be consistent with the nonpine distribution; Z value = 1.96 corresponds to P = 0.05 and Z = 3.89 corresponds to P = 0.0001. Statistical analyses were performed with the SAS software package (SAS Institute, 1999). All P values are two-tailed and statistical significance indicates P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The mineralization of PAHs in forest humus and the self-cleaning potential of the boreal coniferous forest environment for PAH compounds was studied using a model ecosystem with pyrene as the model compound. The model system was a two-dimensional microcosm containing humus from Scots pine forest with or without 4.5-mo-old seedlings of Scots pine, propagated with its mycorrhizal fungus P. involutus. The humus also contained Sphagnum moss and other indigenous vegetation (Fig. 1). Openings of 25 cm2 were cut in the microcosms and refilled with humus containing pyrene (0 or 100 mg kg–1) or with humus–waste oil soil mixture (Fig. 1, Table 1). The implanted microcosms were allowed to equilibrate and then were spiked with [4,5,9,10-14C]-pyrene. Final pyrene concentrations in the implant humus on Day 0 were 0.07, 91.2, or 0.95 mg kg–1, respectively (Table 1).

Figure 3 shows the evolution of 14CO2 from the different microcosms collected as shown in Fig. 2. The 14CO2 emission in the O/0.95 microcosms was considerable already at Day 21. The data in Fig. 3 further show that pyrene was converted to CO2 at rates of 0.6 and 0.07 µg kg–1 d–1 in the O/0.95 and PO/0.95 microcosms, respectively. The rates of pyrene mineralization in the H/91.2 microcosms were initially insignificant but started from Day 64 at the rate of 47 µg kg–1 d–1 and increased to 144 µg kg–1 d–1 after Day 105. In the pine-planted PH/91.2 microcosms the rate also increased, to 28 µg kg–1 d–1, after Day 105. The amount of pyrene emitted as 14CO2 from the PH/0.07 and H/0.07 microcosms containing the lowest amount of pyrene was 0.4 x 10–3 µg kg–1 d–1. The results suggest that the oily soil contained degraders that started to mineralize pyrene soon after spiking. Interestingly, the forest humus, exposed to no PAH source other than airborne distant transport, started emitting 14CO2 from 14C-pyrene within three months, indicating that there were microorganisms in humus with inherent capacity for mineralizing pyrene.



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  		Fig. 3. Cumulative yields of 14CO2 from the two-dimensional microcosms after spiking with 14C-pyrene as described in Table 1. Open circles denote microcosms with Scots pine and Paxillus involutus and an implant of humus plus waste oil soil (PO/0.95); solid circles denote microcosms containing an implant of humus plus waste oil soil (O/0.95); open squares denote microcosms with Scots pine and P. involutus and an implant of humus with pyrene (PH/91.2); solid squares denote microcosms with an implant of humus with pyrene (H/91.2); open triangles denote microcosms with Scots pine and P. involutus and an implant of humus with pyrene (PH/0.07); and solid triangles denote microcosms containing an implant of humus with pyrene (H/0.07). Each line gives the 14CO2 emitted by three parallel microcosms. The bars indicate the standard deviation.

 
In the microcosms with pine, the net evolution of 14CO2 was 5- to 10-fold smaller than in the microcosms without pine (Fig. 3). This was true whether implants contained humus (with 91.2 mg kg–1) or oily soil. To address the question of why the yield of 14CO2 from 14C-pyrene was smaller from the microcosms with than from those without pine the distribution of 14C label on Day 180 inside the microcosms was investigated (Table 2). The data show that part of the 14C label was retained in the pines and also in the ground vegetation. When the vegetation-contained 14C is taken into account, the sum of 14CO2 plus that incorporated by plants represented 12% (PH/91.2) and 9.7% (PO/0.95) of the input. The results indicate that the pines and other vegetation had assimilated 70 to 80% of the 14CO2 generated from the 14C-pyrene. The 14C label accumulated in the ground vegetation was higher in the absence than in the presence of pine, indicating higher 14CO2 assimilation by ground vegetation when the competing pine was absent.

The pathway of incorporation of pyrene 14C label into the pine needles and roots (assimilation of 14CO2 or by absorption of pyrene or its metabolites) was analyzed. Table 3 shows that less than 30% of the total 14C label present in the needles was soluble in chloroform or in methanol and water. A similar distribution of 14C label was obtained with roots. This was similar to the 14C distribution in needles and roots of pines grown with 14C-L-glutamic acid. The fact that the extraction properties and the distribution of the 14C label were similar irrespective of the substrate (L-glutamate or pyrene) indicates that 14C-pyrene label recovered in the pine was due to assimilated 14CO2.


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  		Table 3. Distribution of 14C label in extracts prepared from needles and roots 180 d after spiking the two-dimensional microcosms with 14C-pyrene or 14C-glutamic acid.

 
The 14C label found in the vegetation probably originated from assimilated 14CO2. Taking this into account the gross mineralization rates in the PH/91.2 microcosms were 62 µg kg–1 d–1 and in H/91.2 microcosms 106 µg kg–1 d–1. For the PO/0.95 microcosms the corresponding figure was 0.5 µg kg–1 d–1 and for the O/0.95 microcosms 0.7 µg kg–1 d–1. In the PH/0.07 and H/0.07 microcosms the rates of pyrene mineralization were low (<3 x 10–3 µg kg–1 d–1). The differences in the rates of pyrene mineralization between these three types of microcosms thus reflected the differences of the pyrene concentrations (91.2, 0.95, and 0.07 mg kg–1) (i.e., substrate availability).

The amount of pyrene 14C label recovered as 14CO2 or as 14C labeled ground vegetation was 21% in H/91.2 microcosms and 13% in O/0.95 microcosms in the 6-mo period. Within the nonpine series of experiments, pyrene was degraded more efficiently in humus containing highest amount of pyrene (91.2 mg kg–1) than in the oil-polluted soil, and the difference between H/91.2 and O/0.95 microcosms was statistically significant (P = 0.036) (Table 2). This may relate to the higher pyrene contents of the spiked humus compared with oily soil (91.2 vs. 0.95 mg kg–1), allowing for higher access to pyrene as substrate. Less than 0.5% of the pyrene was mineralized in the PH/0.07 microcosms and in H/0.07 microcosms spiked with a low pyrene content. Apparently the lowest concentration was substrate limiting.

The results (Table 2) allow comparison of pyrene mineralization in microcosms with and without pine. The amount of pyrene recovered as CO2 or found in the vegetation was 10 to 12% with pine and 13 to 21% without pine. More pyrene was degraded in the implants containing the two highest concentrations of pyrene in the nonpine series (H/91.2 and O/0.95) compared with the pine series (PH/91.2 and PO/0.95); the difference being marginally significant in an analysis of variance (P = 0.054). In contrast, in implants containing the lowest amount of pyrene (PH/0.07 vs. H/0.07) more pyrene was degraded in the pine series compared with the nonpine series, but this difference was not statistically significant in the t test (P = 0.2). However, the variance in the pine experiment (PH/0.07) was significantly larger than in the nonpine experiment (H/0.07); the latter providing much more accurate results (Table 2). Therefore, the normal distribution corresponding to the nonpine experiment (H/0.07) and corresponding Z values of the three individual pine replicates of PH/0.07 microcosms were calculated. Two of the pine replicates of PH/0.07 were significantly inconsistent with the nonpine distribution (metabolization = 0.48%, Z = 14; metabolization = 0.68%, Z = 24; P < 0.0001 for both) whereas the third replicate is consistent with the nonpine experiment (0.15%, Z = 1.6, P = 0.44). Thus, if we use the more accurate nonpine experiment (H/0.07) as a basis for comparison, there is strong indication for higher total pyrene metabolization in two of the replicated microcosms of PH/0.07, even though the t test did not find significant overall differences between the means. Accordingly, the results indicate that the presence of growing pine and its mycorrhizal fungus may reduce pyrene degradation in the microcosms with high pyrene concentrations, whereas pine appeared to increase the degradation at the low pyrene concentrations in some pine replicates. Yet the total degradation was low also in those replicates, differing from the nonpine experiment.

Table 2 shows that in most cases, from 70 close to 90% of the 14C-pyrene label remained in the implanted humus. To distinguish between unchanged pyrene and its polar metabolites (other than CO2) the humus was extracted with solvent. According to the results shown (Table 4) around 70% of the pyrene 14C label was extractable in nonpolar solvent (chloroform) and about 30% was nonextractable in all samples regardless the extent of pyrene degradation in that microcosm. Only low amounts of polar metabolites (<2%) partitioning in the methanol and water phase were found (Table 4). Less than 0.1% of the pyrene 14C label was found in the activated carbon (Table 2) designed to trap volatile organic compounds (Fig. 2). This shows that no significant amount of volatile metabolites other than CO2 was formed from pyrene.


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  		Table 4. Extractability of 14C label from 14C-pyrene spiked humus implants inserted in two-dimensional microcosms for 180 d.

 
The sum of 14C label in the humus plus the label accumulated in the plants or volatilized as 14CO2 amounted to 70 to 90% of the input 14C-pyrene. The conclusion is that pyrene, when metabolized, was mineralized to CO2 and then partly assimilated to biomass as compounds insoluble in organic solvents. The amount of polar or volatile metabolites was negligible (<1%).

To compare the biotic and abiotic contributions, mineralization of pyrene was followed in sterile and nonsterile humus. No degradation (<0.1%) was observed in the sterile humus whereas 45% (5.8% SD, n = 3) was mineralized in the natural humus in 170 d. Mineralization of pyrene in humus therefore was biotic implying that humus microbes are capable of pyrene degradation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study the mineralization of pyrene in pine forest humus amended with pyrene or with waste oil soil was examined in microcosms with and without pine and its mycorrhizal fungus. The microcosms were design to resemble the coniferous forest ecosystem by using forest humus and Scots pine infected with mycorrhizal fungus P. involutus. During the development of the microcosms Sphagnum moss started to grow on top and at the sides of the humus layer. This indicates that only these humus regions were exposed to light. The humus layer in the microcosms was thin (5 mm) thus possibly creating a more aerobic environment for the roots than there would be in forest subsurface. The pines were, however, growing well. They were healthy looking and the needles had a dark green coloring which indicated that the pines were not suffering from nutrient deficiency. The pines were actively growing, <1-yr-old seedlings; thus, the results obtained during the study may mainly be valid in forests containing young pine seedlings.

The pine or the fungus did not appear to avoid the contaminated implant region (see the vigorous growth of the fungus at the implant region in Fig. 1a). The size of the contaminated implant was relatively small (1/16 of the size of the total humus layer), which might have helped the pine and the fungus to withstand the contamination. However, the pyrene, even at the highest concentration of 91.2 mg kg–1, seemed not to be toxic to the pine or the fungus.

Pyrene was mineralized most efficiently (>100 µg kg–1 d–1) in microcosms containing pyrene-spiked humus and no pine. Our results showed that the acid conifer forest humus contained a microbial population with an intrinsic potential to mineralize pyrene. Since humus soil is a product formed from lignin and other compounds of plant origin, microbes living in humus may possess a suitable genetic makeup for also degrading polyaromatic compounds of anthropogenic origin.

Polycyclic aromatic hydrocarbons are shown to mineralize in soils with previous history of PAH contamination (see for example Herbes, 1981; Herbes and Schwall, 1978; Klinge et al., 2001; Roper and Pfaender, 2001). We showed here that pyrene is readily mineralized in humus mixed with oil-polluted soil. The oily soil had a >20-yr history of repeated contamination with oil waste and thus probably contained microbes capable of PAH mineralization. We also showed that pyrene is mineralized in pristine forest humus spiked with 91.2 mg kg–1 after a lag period. Roper and Pfaender (2001) have recently described similar results for PAH mineralization in pristine mineral soil from hardwood forest. Thus it seems that at least certain soils with no previous history of PAH contamination can adapt to mineralize PAHs. Indeed, it has been shown that rapid microbial evolution may take place in humus (Sarand et al., 2001).

Acidity usually depresses microbial degradation activity (Hambrick et al., 1980; Kästner et al., 1998; Leahy and Colwell, 1990). The coniferous forest humus, shown in this study to effectively mineralize pyrene, had a pH of 3.6. There appears to be only few reports on pyrene mineralization at low pH. Recently, Roper and Pfaender (2001) showed mineralization of pyrene in mineral soil of hardwood forest at pH 4.8. Grosser et al. (1991) showed pyrene mineralization in soil from a coal gasification plant at pH 4.4. Metabolism, but not necessarily mineralization, of pyrene of sewage sludge origin at a slow rate (t1/2 = 320 d) has been observed in rural coniferous forest soil of pH 2.9 (Wild and Jones, 1993).

The mineralization rates in the Scots pine forest humus reported in this paper ranged from less than 0.4 x 10–3 µg kg–1 d–1 (initial pyrene concentration of 0.07 mg kg–1) to 144 µg kg–1 d–1 (initial pyrene concentration of 91.2 mg kg–1), thus the rates were concentration dependent. The rates were comparable with those reported earlier (0.1 mg kg–1 d–1 with initial concentration of 31 mg kg–1, or 2.7 mg kg–1 d–1 with initial pyrene concentration of 186 mg kg–1) (Carmichael and Pfaender, 1997). If the rates measured in the microcosms are also valid in the forest, the degradation of pyrene contamination of 100 mg kg–1 would take two years when the slow-down of the degradation rate caused by a decreasing substrate concentration or the possible cessation of the degradation during winter months is not considered.

Most of the pyrene-originating 14C label in the microcosm soils remained in solvent (chloroform and methanol) extractable state throughout the study. Only low amounts of 14C label were soluble in the polar (methanol and water) phase. This is similar to the observation for PAHs reported by Herbes and Schwall (1978): when the transformation in freshwater sediment was slow, no oxidized intermediates accumulated. In the present study CO2 and bound 14C were the main products, similar to the study of Herbes (1981) when the transformation of PAHs was followed in sediments. Our observation that no volatile organic metabolites were formed is similar to that reported by Heitkamp and Cerniglia (1987) for PAHs in microcosms containing sediment and water.

The use of plants and their associated microbes has received much attention in soil cleaning, because some pollutants have been reported to degrade faster in the rhizosphere than in unvegetated soil (Anderson et al., 1993; Aprill and Sims, 1990; Banks et al., 1999; Boyle and Shann, 1998, Miya and Firestone, 2000; Reilley et al., 1996). Three different species of pines were recently shown to promote degradation but not necessarily mineralization of pyrene in silt loam (pH = 6.3) (Liste and Alexander, 2000). Recent laboratory experiments with clover (Trifolium repens L. cv. Grasslands huia) and ryegrass (Lolium perenne L. cv. Barclay) have shown that dissipation of anthracene, chrysene, and dibenz(a,h) anthracene may be enhanced in the presence of arbuscular mycorrhiza (Joner et al., 2001). A fungal strain belonging to the same species as that used in the present study, P. involutus, converted pyrene in liquid culture under nonsymbiotic growth conditions (Gramss et al., 1999). We observed no positive effect on pyrene mineralization by the Scots pine and its mycorrhizal fungus P. involutus in microcosms containing pyrene (91.2 or 0.95 mg kg–1). Rather, the pyrene degradation was reduced in the presence of pine and mycorrhizal fungus.

A positive (although statistically not significant) effect of pine and fungus on the pyrene degradation was seen in the microcosms containing the lowest amount of pyrene (0.07 mg kg–1). This result is comparable with the trend found in earlier studies. It has been shown that the effect of vegetation on the degradation of organic compound was more apparent at conditions where the bioavailability of a compound was low or the concentration of a compound had decreased to a level where the microorganisms were no longer able to degrade the compound efficiently (Hutchinson et al., 2001; Reilley et al., 1996).

The efficient degradation of pyrene in humus (with no roots) may indicate that at the highest pyrene concentration (91.2 mg kg–1) the humus (without roots) contained enough substrate to allow microbes to degrade pyrene efficiently. In the microcosms that contained oily soil, there was high concentration of petroleum hydrocarbons. These compounds may have functioned as a C source for the microbes thus possibly allowing the microorganisms to degrade pyrene cometabolically. The duration of the present study was relatively short (half a year). According to Hutchinson et al. (2001) the total petroleum hydrocarbons declined with no significant differences between vegetated and unvegetated treatments during the first 6 mo but the total organic C degradation was significantly greater in vegetated treatments compared with unvegetated treatments after one year when the effect of the soil processing had been overcome.

The fastest degradation rate obtained in this study was equivalent to 50 mg kg–1 yr–1 (initial pyrene concentration of 91.2 mg kg–1) and the slowest approximately 1 µg kg–1 yr–1 (initial pyrene concentration of 0.07 mg kg–1). In southern Finland the total annual PAH deposition is about 0.1 mg m–2 yr–1 (Korhonen et al., 1998). If 1 m2 of forest soil has a 0.5-cm layer of humus weighing approximately 1.5 kg the annual deposition would be equivalent to 0.07 mg PAH kg–1 humus. The measured mineralization rates indicate that the pyrene is unlikely to accumulate in the Finnish forest environment at the present deposition rate. Local elevation of pyrene concentration may increase the degradation capacity and lead to faster degradation in forest humus.


   ACKNOWLEDGMENTS
 
We thank Robin Sen (University of Helsinki) for providing the fungus P. involutus, Sari Timonen for help with setting up the two-dimensional microcosms, Roger Hurme for help with the radioisotope analysis, and Harri Hemilä for help with the statistical analysis. Annele Hatakka and Martin Hofrichter are thanked for helpful discussions and Jarmo Juuti for the help with image processing. The work was supported by several grants from the Academy of Finland to Martin Romantschuk and Mirja Salkinoja-Salonen (SA 52798).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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