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ARTICLE IN PRESS International Biodeterioration & Biodegradation 58 (2006) 15–21 www.elsevier.com/locate/ibiod Biodegradation of polycyclic aromatic hydrocarbons in forest and salt marsh soils by white-rot fungi L. Valentı´ na,1, G. Feijooa, M.T. Moreirab,Ã, J.M. Lemab a Department of Chemical Engineering, Institute of Technology, University of Santiago de Compostela, E-15782 Santiago, Spain b Department of Chemical Engineering, School of Engineering, University of Santiago de Compostela, E-15782 Santiago, Spain Received 15 November 2005; received in revised form 31 March 2006; accepted 11 April 2006 Available online 26 May 2006 Abstract Single strains of nine white-rot fungal species were screened for the ability to degrade polycyclic aromatic hydrocarbons (PAHs) in forest and salt marsh soils. Of these, Bjerkandera adusta, Irpex lacteus and Lentinus tigrinus were outstanding PAH degraders in both saline and non-saline conditions. These three strains were tested in parallel to investigate the levels of salinity and PAH concentration which had negative effects on the ligninolytic activity of the fungi as measured by the anthraquinone dye Poly R-487. Salinity characteristic of sea water had minimal effect on ligninolytic activity of I. lacteus and L. tigrinus, while in B. adusta, activity was inhibited by salinity levels of 32%. The results also indicated that the concentration of each single PAH should be o10 mg lÀ1, equivalent to a concentration of 100 mg kgÀ1 polluted soil. These results were statistically analysed using analyses of covariance (ANCOVA). The statistical analyses conﬁrmed that the decolourization rate varied with salinity and PAH concentration, and between fungi. In the light of these results, future research should be focused on the selection of the most appropriate bioreactor conﬁguration for soil detoxiﬁcation. r 2006 Elsevier Ltd. All rights reserved. Keywords: Biodegradation; Polycyclic aromatic hydrocarbons (PAHs); Soil-slurry system; White-rot fungi; Marsh soil; Forest soil; Salinity 1. Introduction compounds (Bumpus, 1989; Field et al., 1992; Barr and Aust, 1994; Bogan and Lamar, 1996; Pointing, 2001). Polycyclic aromatic hydrocarbons (PAHs) are important The natural environment of these fungi is wood, in which pollutants of soil and sediments. Most biological ap- they grow, secreting ligninolytic enzymes that depolymerize proaches considered for the restoration of PAH-contami- the lignin. A major drawback arises when these fungi are nated sites depend on the activity of bacteria. A possible intended for remediating polluted soils, where the promo- alternative studied for degradation of this type of tion of their growth and generation of ligninolytic enzymes compound is the use of ligninolytic fungi. These possess have been found to be difﬁcult (Okeke and Agbo, 1996). an extracellular degradation system which is capable of Moreover, PAHs have a strong tendency to sorb onto the breaking down lignin (Kirk and Farrell, 1987), an soil matrix, and thus their desorption from soil limits their amorphous and complex biopolymer with an aromatic availability for fungal biodegradation. structure similar to the aromatic molecular structure of Very few reports have considered the complexity of some environmental pollutants such as PAHs, pesticides, degradation of PAHs in soil-slurry systems by white-rot polychlorinated biphenyls (PCBs), synthetic dyes, etc. This fungal cultures (Brodkorb and Legge, 1992; Zheng and structural resemblance makes possible the use of white-rot Obbard, 2002). Even more, there is still a lack of fungi to treat sites contaminated with these recalcitrant information about fungal growth and PAH oxidation in soil-slurry reactors treating soil from marine areas. Such ÃCorresponding author. Fax: +34981528050. information is basic for the successful treatment of oil spills E-mail address: firstname.lastname@example.org (M.T. Moreira). at shorelines and marsh ecosystems. Essential aspects to be 1 Present address: Department of Applied Chemistry and Microbiology, considered for application of these fungi to detoxiﬁcation University of Helsinki, 00014 Helsinki, Finland. of marine sites contaminated with PAHs are (a) selection of 0964-8305/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2006.04.002 ARTICLE IN PRESS 16 ´ L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21 fungal strains with high degradative ability and (b) Table 1 evaluation of the effects of the high salinity associated Physico-chemical characterization of forest and marsh soils with coastal areas on fungal growth, production of Forest soil Marsh soil enzymes and ligninolytic activity. The ligninolytic potential of these fungi is related to the secretion of oxidative pH 4.04 5.57 enzymes, which permit the effective decolourization of an Carbon concentration 7.85% 2.12% Nitrogen concentration 0.55% 0.55% anthraquinone dye, Poly R-478 (Glenn and Gold, 1983). Organic matter 9.8 1.6–2.2 The main purpose of this study was to screen nine well- CEC (cmolc kgÀ1)a 4.98 115.53 known lignin-degrading white-rot fungi suitable for treat- Mg2+ (cmol kgÀ1) 0.73 98.40 ing salt marsh soil polluted with PAHs, focusing on four Na+ (cmol kgÀ1) 0.04 13.05 different PAHs with three (phenanthrene (PHEN)) or four Ca2+ (cmol kgÀ1) 2.5 2.91 K+ (cmol kgÀ1) 0.2 1.17 benzene rings (ﬂuoranthene (FLU), pyrene (PYR) and Al3+ (cmol kgÀ1) 1.51 0.11 chrysene (CHRY)). An initial screening assay was con- Fe (g kgÀ1) 15.9 1.71 ducted on forest and marsh soils under non-saline and Mn (g kgÀ1) 30 184 saline conditions, respectively, with the aim of selecting Zn (g kgÀ1) 149 80 those strains with better degradative potential in both a CEC: cationic exchange capacity, deﬁned as the amount of exchange- cases. Thereafter, three fungi selected on the basis of these able cations that can be adsorbed onto the soil at pH 7. assays were studied in more detail to assess levels of sea water content and PAH concentration having minimal effect on dye decolourizing capacity as a measurement of Table 2 ligninolytic activity. This work is part of a wider project for Properties and initial concentration of PAHs in forest and marsh soils development of remediation technology based on the use of (based on dried weight of soil) a soil-slurry reactor for the degradation of PAHs in marine PAH compound Number Solubility Ionization Concentration areas, in order to assess its applicability in treating oil of rings (mg lÀ1)a potential (eV)b (mg kgÀ1) spills. Phenanthrene 3 1.29 8.03 50 Fluoranthene 4 0.26 7.95 50 2. Materials and methods Pyrene 4 0.14 7.41 50 Chrysene 4 0.002 7.60 50 2.1. Microorganisms a Sims and Overcash (1983). b Levin and Lias (1982). The fungal strains screened were Phanerochaete chrysosporium BKM- F-1767, Phanerochaete sordida YK-624, Polyporus ciliatus ONO94-1, Stereum hirsutum PW93-4, Lentinus tigrinus PW94-2, Bjerkandera adusta coast (Spain). Each soil was sieved and the fraction passing a 0.5-mm mesh BOS55, Irpex lacteus Fr. 238 617/93, Pleurotus eryngii CBS 613.91 and stored at 4 1C. Prior to use, this soil was autoclaved (121 1C, 20 min) and Phlebia radiata WIJSTER94-6. All strains were from the culture collection spiked with PAH in acetone. The solution contained PHEN, FLU, PYR of the Chemical Engineering Department, University of Santiago de and CHRY to achieve a ﬁnal individual PAH concentration in the soil of Compostela (Spain). All were transferred from culture ﬂasks (maintained 50 mg kgÀ1 soil (Table 2). The soil was then held for 24 h under a fume at 4 1C) to malt extract agar plates comprising (lÀ1) 15.0 g agar, 3.5 g malt hood for the solvent to evaporate. extract and 10.0 g glucose, and incubated at 30 1C for 5–7 days before being stored at 4 1C and subcultured every 6 months. 2.4. Screening assays 2.2. Preparation of fungal inoculum Small-scale screening was performed in 100-ml Erlenmeyer ﬂasks Fernsbach ﬂasks (1 l) containing 100 ml modiﬁed Kirk medium (Tien containing a slurry of 2 g (dry weight) soil in 16 ml culture medium and Kirk, 1988) were each inoculated with ﬁve 5-mm plugs of active prepared with distilled water and BIII mineral medium for the forest soil, mycelium cut from malt extract agar plate cultures, and incubated at and with marsh water for the marsh soil (Table 3), to which 4 ml inoculum 30 1C. The culture medium contained (lÀ1) 10 g glucose as carbon source, was added to give an initial fungal biomass of 1.4 g lÀ1 (dry weight). The 2 g ammonium tartrate or peptone as nitrogen source and 100 ml BIII ﬂasks were closed with a cotton plug and incubated at 30 1C in total mineral medium. Prior to sterilization by autoclaving, the medium was darkness for 30 days in an orbital shaker operating at 120 rpm to gently adjusted to pH 4.5 with 20 mM acetate buffer. After autoclaving, ﬁlter- agitate the soil. Six replicates of abiotic controls were harvested after 1 and sterilized thiamine stock solution (200 mg lÀ1) was added at the rate of 30 days, and four replicates for each fungus harvested after 30 days in 1 ml lÀ1 medium. After 7 days, the contents of each culture ﬂask were order to measure the residual PAHs in the soil. homogenized in a sterilized blender for 1 min, providing inoculum for the screening assays. 2.5. Effect of salinity and PAH concentration on dye decolourizing rate 2.3. Soil preparation The effects of salinity and PAH concentration on decolourization of Forest soil and marsh soil with characteristics determined by standard Poly R-478 dye, a polyanthraquinone (MW ¼ 220–250 kDa) which has a methods (ASTM, 1999) (Table 1) were used for the screening assays. The structural resemblance to some PAHs, were analysed using B. adusta, I. forest soil was collected at a depth of 20 cm from a grassy area in an oak lacteus and L. tigrinus. The decolourization capacity served as an indirect grove and the marsh soil was harvested from a salt marsh on the Galician measurement of the enzyme activity of each fungus (Gold et al., 1988). ARTICLE IN PRESS ´ L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21 17 Table 3 3. Results and discussion Chemical composition of marsh and sea water Compounds Marsh water Sea water 3.1. Screening in forest soil Mg2+ (mg lÀ1) 525 1270 The biodegradative and abiotic losses of PAHs with time Na+ (mg lÀ1) 4000 10710 of incubation for the forest soil are summarized in Table 4. Ca2+ (mg lÀ1) 188.4 400 The data presented, corresponding to the average value of K+ (mg lÀ1) 253.4 390 Mn+ (mg lÀ1) 0.09 0.05 six parallel controls taken at zero time and day 30 and NO3À (mg lÀ1) 0.7 1.24 samples in triplicate at day 30, show that all fungi except PO3(mg lÀ1) 4 0.062 0.088 Phlebia radiata were capable of degrading the mixture of Salinity (%) 10 32 PAHs to some extent, although Pleurotus eryngii, B. adusta and I. lacteus were outstanding strains with average degradation of the four PAHs being 67%, 55% and Decolourization assays were performed statically in 100-ml Erlenmeyer 53%, respectively, by the three strains. ﬂasks containing 15 ml culture medium comprising (lÀ1) 10 g glucose, The recovery of the PAHs from incubated biologically 100 ml BIII mineral medium and 0.2 g Poly R, and adjusted to pH 4.5 and amended with thiamine as described earlier. For examination of the effect inactive control soils was different for each PAH. The of salinity, the medium was made up in distilled water and 25%, 50%, coefﬁcients of variation between controls at zero time and 75% and 100% sea water (Table 3), and for the effect of PAH, it was made day 30 did not exceed a maximum of 20%, except for up in distilled water with each individual PAH added concentrations of PHEN for which degradation was somewhat obscured by zero, 1, 5, 10 and 50 mg lÀ1. These were inoculated with two 5-mm mycelial strong abiotic losses, primarily by volatilization during plugs and incubated under air at 30 1C for 22 days. The dye was added to give a concentration of 0.2 g lÀ1 culture medium immediately before incubation. This effect was evident for samples taken at incubation (zero time). Decolourization of the dye was monitored as day 30, when the loss in PAH concentration ranged from percentage reduction in the absorbance ratio at 520 and 350 nm in 35% to 45%. However, it was still possible to detect comparison with the dye control. Before reading absorbance, 0.2 ml signiﬁcant divergence between controls and biologically centrifuged sample (20,000 Â g, 10 min) was diluted with 0.8 ml distilled active samples, with an average difference around 30–50%. water and measured spectrophotometrically at 30 1C in a 1-cm cuvette. Experimental assays and controls were run in triplicate. All strains screened were able to degrade FLU, with degradation ranging from 40% (Phanerochaete chrysospor- ium) to 71% (Pleurotus eryngii), except for Phlebia radiata, 2.6. Extraction and analyses which degraded o12%. More than 54% of FLU was degraded by fungi such as I. lacteus, S. hirsutum and B. All culture ﬂasks were weighed before and after the experiment to correct for concentration effects resulting from evaporation of water. The adusta. Less active species such as Phanerochaete sordida, whole assays were extracted in order to analyse the residual PAH in the Polyporus ciliatus, Phanerochaete chrysosporium and L. soil. Hexane–acetone (1:1, 40 ml) was added to each ﬂask and agitated for tigrinus achieved degration rates up to 48%. PYR was the 2 h at 300 rpm in a horizontal shaker in order to achieve the transport of most extensively degraded compound with 85%, 83% and PAHs from water or soil to the organic solvent phase. Following this, if an 81% being degraded by, respectively, I. lacteus, Phaner- emulsion formed during agitation, the contents were sonicated for a further 30 min for separation of the organic solvent and aqueous phases. A ochaete sordida and B. adusta. However, Pleurotus eryngii 1.5-ml aliquot of the liquid was ﬁltered through nylon to remove small soil particles and collected in an amber vial. The average extraction efﬁciencies Table 4 for forest soil were 88.92712.14% (PHEN), 95.2777.75% (FLU), Residual PAH concentration (mg kgÀ1) after incubation of nine strains of 93.5477.59% (PYR) and 90.6876.78% (CHRY). The corresponding white-rot fungi for 30 days under non-saline conditions (forest soil) average efﬁciencies for marsh soil were 93.4374.92%, 97.1474.47%, 75.7973.29% and 81.9373.55%. The contents of the vials were analysed Phenanthrene Fluoranthene Pyrene Chrysene on a Hewlett-Packard HP 1090 HPLC equipped with a diode array detector monitoring the absorbance at 235 nm for PHEN and FLU, Control, zero 45.076.1 47.473.9 46.873.8 45.373.4 240 nm for PYR and 254 nm for CHRY. A 4.6 Â 200 mm Spherisorb time ODS2 reverse-phase column (5 mm; Waters) and a HP ChemStation data Control, 30 days 30.472.9 36.673.7 35.173.4 42.2710 processor were used for determining the PAH concentration. The injection Phanerochaete 20.371.7 22.071.6 14.172.5 23.570.2 volume was set at 10 ml and the isocratic eluent (acetonitrile–water, 80:20) chrysosporium was delivered at a rate of 0.4 ml minÀ1. Phanerochaete 19.972.8 19.273.0 6.072.1 32.371.8 sordida Polyporus 20.471.6 20.372.5 12.270.9 32.071.6 2.7. Statistical analysis ciliatus Stereum 17.171.4 16.272.4 13.470.1 31.972.1 The effects of salinity and PAH on the decolourization of Poly R-478 hirsutum were evaluated by covariance analysis (ANCOVA). Each of the two Lentinus tigrinus 17.873.1 21.871.7 14.172.7 31.171.0 experiments was considered as a factorial design consisting of four Bjerkandera 14.470.9 16.971.2 6.570.9 28.772.3 treatments (the three fungal species and the control) with three replicates adusta per treatment and two covariates, time and either PAH or salinity to Irpex lacteus 18.871.3 15.771.7 5.171.6 29.271.2 deﬁne the covariation of either salinity or PAH and species with time (i.e. Pleurotus eryngii 2.070.3 10.577.3 9.678.3 29.072.4 the rate of decolourization). All analyses were carried out in statistical Phlebia radiata 25.771.6 32.473.9 30.073.2 34.073.4 package R. ARTICLE IN PRESS 18 ´ L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21 (73%) was also an outstanding PYR degrader, and except Table 6 for Phlebia radiata (15%), the remaining fungi were able to Average net PAH degradation (%) for Bjerkandera adusta, Irpex lacteus and Lentinus tigrinus under saline (marsh soil) and non-saline conditions convert 460% of the PYR. CHRY was degraded least, the (forest soil) average net degradation rate being 28%, with Phaner- ochaete chrysosporium degrading 45% and B. adusta, I. Phenanthrene Fluoranthene Pyrene Chrysene lacteus and Pleurotus eryngii 31–32%. The expectation of Marsh soil 48 51 51 49 an inverse correlation between the number of PAH rings Forest soil 44 50 76 30 and the loss from soils was supported by the results, with the loss for the 3-ring PAH, PHEN, from the forest soil (and also the marsh soil) being somewhat less than for the 4-ring PAHs, FLU, PYR and CHRY. Comparatively, the results presented here show greater PAH degradation than obtained for other well-known 3.2. Screening in marsh soil white-rot strains, such as Phanerochaete chrysosporium cultured in soil-slurry systems (Zheng and Obbard, 2002). In the parallel assays performed for salt marsh soil using This strain showed lower percentage degradation of PHEN marsh water in the culture medium (Table 5), a strong (29%), FLU (16.4%) and PYR (21.9%), and no CHRY inhibitory effect on PAH degradation was evident except degradation, in the presence of 11 mM Mn2+. The results for three fungi, viz. I. lacteus, B. adusta and L. tigrinus, from the present screening tests revealed that B. adusta, I. which on average degraded the four PAHs by 57%, 49% lacteus and L. tigrinus were outstanding PAH degraders, in and 42%, respectively. Four species (L. tigrinus, B.adusta, both saline and non-saline conditions, with average I. lacteus and Pleurotus eryngii) degraded 440% of the degradation of 48% and 44% (PHEN), 51% and 50% PHEN, o22% was degraded by Phanerochaete chrysos- (FLU), 51% and 76% (PYR) and 49% and 30% (CHRY) porium, Polyporus ciliatus and S. hirsutum. The greatest in the presence of 1.36 (marsh) and 33 mM Mn2+ (forest), FLU and PYR degrader was I. lacteus, which degraded, respectively (Table 6). respectively, 60% and 63% in 30 days under saline An important criterion for the selection of PAHs which conditions. B. adusta and L. tigrinus also notable, may be effectively degraded by the ligninolytic enzymes is degrading 54% and 39% of FLU and 49% and 40% of the ionization potential, IP (Table 2). Among ligninolytic PYR, respectively. These three fungi also showed the enzymes, lignin peroxidase (LIP) can degrade PAHs with greatest ability to degrade CHRY (56%, 48% and 43%, IP o7.55 eV (Hammel et al., 1985), and manganese respectively). In contrast, low FLU and PYR degradation peroxidase (MnP) with IP o7.8 eV (Cavalieri and Rogan, rates were shown by Phlebia radiata (5% and 3%, 1985). Theoretically, the enzymatic system should be respectively) and Phanerochaete chrysosporium (8% and efﬁcient provided that the oxidation potential of a 12%, respectively). No CHRY degradation was detected particular compound is lower than that supplied by the for S. hirsutum and only negligible amonts removed by enzymic cycle. Among the tetracyclic PAHs, the most Pleurotus eryngii (9%) and Phanerochaete chrysosporium condensed, PYR (IP ¼ 7.41 eV), which lacks a bay region, (12%). was subject to the most rapid biodegradation. Moreover, the soil-slurry system proved to oxidize PHEN and FLU, Table 5 which have IP values 47.8 eV. Therefore, it is worthwhile Residual PAH concentration (mg kgÀ1) after incubation of nine strains of mentioning the very marked ability of these fungi and their white-rot fungi for 30 days under saline conditions (marsh soil) associated oxidative enzymes to degrade PAHs. One hypothesis to account for this could be that the extra- Phenantrhene Fluoranthene Pyrene Chrysene cellular system secretes other compounds participating in Control, zero 46.772.5 48.672.2 37.971.6 41.071.7 the degradative action of the enzymes. Among the different time possibilities, lipid peroxidation may be the catalytic Control, 30 days 25.6711 38.076.0 38.376.7 38.376.7 mechanism for the oxidation of PAHs, as described for Phanerochaete 23.072.1 35.174.4 33.772.8 33.874.5 PHEN (Moen and Hammel, 1994). chrysosporium Phanerochaete 16.874.1 29.672.7 30.571.3 30.872.7 sordida 3.3. Effect of saline conditions and PAH concentration on Polyporus 20.372.4 28.772.1 28.171.0 32.972.3 fungal ligninolytic activity ciliatus Stereum 24.070.6 32.471.1 31.672.5 45.271.6 The evaluation of the possible inhibitory effect of salinity hirsutum Lentinus tigrinus 13.970.7 23.170.7 22.871.0 21.870.9 is essential for the selection of the most suitable strains for Bjerkandera 13.371.0 17.770.7 19.771.2 20.173.7 the remediation of shorelines and coastal areas polluted adusta with oil spills. The concentrations of salts in the medium Irpex lacteus 13.072.4 15.170.8 14.171.6 16.770.1 and the PAH concentrations being major factors that could Pleurotus eryngii 13.272.4 28.470.7 32.071.5 34.970.3 affect degradative capacity, the objective of our experi- Phlebia radiata 16.671.0 36.270.6 37.270.8 29.971.7 ments was to determine the levels of salinity and PAH ARTICLE IN PRESS ´ L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21 19 concentration which affect the degradative capability tion between time and species, F3, 538 ¼ 99.77, Po0:001). of the three selected fungi, B. adusta, I. lacteus and L. The response of different fungi to salinity was also variable tigrinus. (ANCOVA, three-way interaction between time, salinity As mentioned in Section 2, the effect of concentration of and species, F3, 538 ¼ 24.31, Po0:001). Overall, the the salts was determined in a ﬁrst experiment where assays presence of sea water (which has average salinity values were performed with different proportions of sea water in of around three-fold higher than in marsh water) had no the culture medium. It can be seen from the dye detrimental effect on the rate of decolourization for either decolourization assay (Fig. 1), used to assess the extra- I. lacteus or L. tigrinus, but for B. adusta decolourization cellular ligninolytic enzyme activity because fungal bior- was inhibited by 100% sea water (Fig. 1). Therefore, emediation potential is often attributed to these enzymes, although the salinity experiments indicate that these fungi that decolourization started after an initial lag of approx. 5 can perform the degradation in a soil from a marine days and continued throughout the 22 days of the environment, the employment of this strain of B. adusta experiment. The statistical analysis (ANCOVA) corrobo- would require control of the salt content to prevent rated that time had an effect on the decolourization (F1, inhibition. Having in mind that this can be an ex situ 538 ¼ 1093.02, Po0:001). On the other hand, the decolour- technique, preconditioning of the soil to remove ization rate varied with sea water content (ANCOVA, salts would not be necessary, although it would not be interaction between time and salinity, F1, 538 ¼ 3.87, advisable to use sea water for inoculum and culture media Po0:05) and between fungal species (ANCOVA, interac- preparation. 1.2 1.2 1.0 1.0 0.8 0.8 A520/A350 A520/A350 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 5 10 15 20 25 0 5 10 15 20 25 (A) Time (days) (B) Time (days) 1.2 1.2 1.0 1.0 0.8 0.8 A520/A350 A520/A350 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 5 10 15 20 25 0 5 10 15 20 25 (C) Time (days) (D) Time (days) 1.2 1.0 0.8 A520/A350 0.6 0.4 0.2 0.0 0 5 10 15 20 25 (E) Time (days) Fig. 1. Decolourization rates of the dye Poly R-478 by the white-rot fungi Lentinus tigrinus ( Â ), Bjerkandera adusta (&) and Irpex lacteus (m) incubated in culture media prepared with different proportions of sea water: (A) 100%, (B) 75%, (C) 50%, (D) 25% and (E) 0%. ARTICLE IN PRESS 20 ´ L. Valentın et al. / International Biodeterioration & Biodegradation 58 (2006) 15–21 1.2 1.2 1.0 1.0 0.8 0.8 A520/A350 A520/A350 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 5 10 15 20 25 0 5 10 15 20 25 (A) Time (days) (B) Time (days) 1.2 1.2 1.0 1.0 0.8 0.8 A520/A350 A520/A350 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 5 10 15 20 25 0 5 10 15 20 25 (C) Time (days) (D) Time (days) 1.2 1.0 0.8 A520/A350 0.6 0.4 0.2 0.0 0 5 10 15 20 25 (E) Time (days) Fig. 2. Decolourization rates of the dye Poly R-478 by the white-rot fungi Lentinus tigrinus ( Â ), Bjerkandera adusta (&) and Irpex lacteus (m) incubated in culture media containing different PAHs concentrations (mg lÀ1): (A) 50, (B) 10, (C) 5, (D) 1 and (E) 0. In the second experiment evaluating the effect of PAHs Acknowledgements on dye decolourization, the effect of time on the levels of absorbance ratio (A520/A350) was signiﬁcant, conﬁrming This work was funded by the Spanish Commission of decolourization with time (ANCOVA, F1,587 ¼ 1200.33, Science and Technology (CICYT; Project VEM2003- Po0:001). The effect of PAHs on the fungi (Fig. 2) was to 20089-CO2-01). progressively slow decolourization by all three strains with increasing concentration up to 50 mg lÀ1. (ANCOVA, References interaction between time and PAH, F1,588 ¼ 243.01, Po0:001). Different species showed different rates of ASTM, 1999. American Society for Testing and Materials. Methods: decolourization (ANCOVA, interaction between time and D422-63, D2974-87, D4972-95a, D3974-81. Barr, D.P., Aust, S.D., 1994. Mechanisms white-rot fungi use to degrade species, F3,588 ¼ 62.80, Po0:001) and reacted differently to pollutants. Environmental Science and Technology 28, 78A–87A. 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