ELSEVIER P I I : S 0 2 6 9 - 7 4 9 1 ( 9 6 ) 0 0 0 4 4 - 9 Environmental Pollution, Vol. 94, No. 3, pp. 355 362, 1996 © 1997 Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0269-7491/96 $15.00 + 0.00 B I O D E G R A D A T I O N OF ALIPHATIC A N D AROMATIC H Y D R O C A R B O N S BY N A T U R A L SOIL MICROFLORA A N D PURE CULTURES OF IMPERFECT A N D LIGNOLITIC FUNGI Juan C. Colombo, a* Mar t a Cabello b* & Ang61ica M. Arambar r i b~ "Environmental Chemistry Chair, Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, Paseo del Bosque s/n, La Plata 1900, Buenos Aires, Argentina hInstituto de Botdnica "C. Spegazzini", Facultad de Ciencias Naturales y Museo, Universidad Nacional de La Plata, calle 53 no. 477, La Plata 1900, Buenos Aires, Argentina (Received 2 July 1995; accepted 1 April 1996) Abstract The biodegradation of aliphatic and aromatic hydro- carbons by natural soil microflora and seven fungi species, including imperfect strains and higher level lignolitic species, is compared in a 90-day laboratory experiment using a natural, not-fertilized soil contaminated with 10% crude oil. The natural microbial soil assemblage isolated from an urban forest area was unable to significantly degrade crude oil, whereas pure fungi cultures effectively reduced the residues by 26-35% in 90 days. Normal alkanes were almost completely degraded in the first 15 days, whereas aromatic compounds (phenanthrene and methylphenanthrenes) exhibited slower kinetics. Aspergillus terreus and Fusarium solani, isolated from oil-polluted areas, produced the more efficient attack of aliphatic and aromatic hydrocarbons, respectively. Overall, imperfect fungi isolated from polluted soils showed a somewhat higher efficiency, but the performance of unadapted, indigenous, lignolitic fungi was comparable, and all three species, Pleurotus ostreatus, Trametes villosus and Coriolopsis rigida, effectively degraded aliphatic and aromatic components. The simultaneous, multivariate analysis of 22 parameters allowed the eluci- dation of a clear reactivity trend of the oil components during biodegradation: lower molecular weight n-alkanes > phenanthrene > 3-2-methylphenanthrenes > intermediate chain length n-alkanes > longer chain length n-alkanes > isoprenoids ~9-1-methylphenanthrenes. Irrespective of the individual degrading capacities, all fungi species tested seem to .follow this decomposition sequence. © 1997 Published by Elsevier Science Ltd. All rights reserved INTRODUCTION Modern society industrial activities produce massive amounts of xenobiotic compounds, which, in a high pro- *To whom correspondence should be addressed. +MC is a member of Carrera del Investigador Cientifico de la CIC, Provincia de Buenos Aires. ~AMA is member of Carrera del lnvestigador Cientifico del CONICET, Argentina. 355 portion, finalize their cycle by entering the environment. Thus, aquatic, sediment or soil microbial communities are exposed to a continuously growing set of diverse syn- thetic chemicals (Kobayashi & Rittmann, 1982). As these microorganisms are responsible for the most important environmental process of pollutant disappearance, i.e. biodegradation, in recent years much attention has been devoted to their study, specially focusing on optimizing their degradation potential for bioremediation purposes (Lindstrom et al., 1991; Madsen, 1991). The metabolic and enzymatic diversity and bio- degradation capabilities of bacteria for anthropogenic compounds, including hydrocarbons, chlorobenzenes, polychlorinated biphenyls and other xenobiotics, have been recognized (Atlas, 1981 and references therein; Fava, 1991; Alder et al., 1993; Ye et aL, 1996). However, fungi have also been the subject of recent research because of their ability to synthesize relatively unspecific enzymes involved in cellulose and lignin decay (peroxidases, laccases) that are capable of degrading high-molecular- weight, complex or more recalcitrant toxic compounds, including aromatic structures (Milstein et al., 1988; Katayama & Matsumura, 1991; Hofrichter et al., 1993; Lamar et al., 1993; Sack & Giinther, 1993). In this study, the degradation of aliphatic and aromatic hydrocarbons by a natural soil microbial assemblage and seven fungi species, including imperfect strains and higher level lignolitic species, is compared in a 90-day laboratory incubation experiment with a natural soil contaminated with 10% crude oil. MATERIALS AND METHODS Fungi strain isolation The autochthonous Hyphomycetes fungi were isolated from long-term hydrocarbon-polluted soils using the soil-washing technique (Parkinson & Williams, 1961): Aspergillus terreus Thom LPS 492 from a gas-oil- polluted soil from Mar de Ajo and Fusarium solani (Martius) Sac. LPS 493 and Trichoderma harzianum Rifai LPS 494 from a crude-oil-polluted soil from La 356 J.C. Colombo et al. Plata YPF refinery, both in the Buenos Aires Province. These strains were maintained on Czapex with 1% crude oil at room temperature. The other Hyphomycete tested (Penicillium chrysogenum Thorn LPS 495) was a contam- inating agent of the crude oil used for the experiments that developed only in the sterilized non-inoculated soil sample. The three species of lignolitic Basidiomycetes were Coriolopsis rigida (Berk. et Mont.) Murill. LPS 232 and Trametes villosa (Fr.) Karst. LPS 233 isolated from rotten wood collected in Misiones Province tropical rain forests using standardized basidiomycete culture techni- ques (Ibafiez, 1995), and Pleurotus ostreatus (Jacquin: Fr.) Kunner MUCL 28782. These strains were maintained on 2% malt extract agar. Soil treatment and inoculation The soil and associated microflora used in the experi- ments were sampled in a woodland area of La Plata city with no history of heavy oil pollution and a balanced nutrient status (organic matter, 11.6%; C, 6.7%, N, 0.52%, P, 16 ppm, K, meq/100 g). The soil, collected in pre-cleaned glass jars, was air-dried (approx. 1 week), powdered and sieved through a 2-mm-mesh sieve and then subjected to steam-sterilization (1 h at 120°C repeated three times after a 24-h interval), except one aliquot that was separated to preserve the indigenous soil microflora. The bulk soil was then contaminated with 10% crude oil obtained from La Plata YPF refinery and thoroughly homogenized under sterile conditions (in the same bag used for the steam-sterilization under a laminar-flow bench). The axenic condition of the soil was tested through the incubation of soil particles (7 days at 28°C in malt agar extract). None of the incubated Petri dishes showed signs of microbial development. Biodegradation experiments were carried out in eight 4-1itre sterilized glass flasks (30 min at 120°C, P= 1 kg) loaded under the laminar-flow bench with 1 kg of the bulk contaminated soil. Two control flasks received no inoculation, one preserving the indigenous soil microflora and another sterilized (which was colonized by Penicillium chrysogenum introduced with the oil). For the inocula- tion of Hyphomycetes, the strains were maintained in liquid salt media (Czapex with 10% sucrose) at 28°C in a culture camera for 1 week. The heavily sporulated mycelia was mixed with 100 ml sterilized distilled water and then poured into the flasks under sterile conditions and continuous homogenization. Basidiomycetes grown axenically on Populus wood sticks (2x10 cm) were directly inoculated by introducing 10 sticks down to 8 cm depth in each flask under the laminar-flow bench. The flasks, covered with sterilized paper and aluminium foil to minimize hydrocarbon volatilization, were main- tained under daylight conditions at 25+3°C for 90 days. Samples of 3-5 g were taken after a thorough homogenization in the laminar-flow bench at days 0, 15, 30, 60 and 90. Soil colonization efficiency One aliquot of the sample collected at day 90 was separated for biological analysis. These samples were A C 15 17 19 20 Lyt A 21 I L 26272829303, J 0 10 20 30 40 Retention time (rain) Fig. 1. Gas chromatogram of the aliphatic fraction of the initial 10% oil-polluted soil (A) and the final decomposition stages (90 days) of the natural soil microflora (B) and Aspergillus terreus (C) treatments. Numbers 15-31 correspond to n-alkanes, and Npr, Pris and Phyt stand for the isoprenoids, norpristane, pristane and phytane. processed using the previously cited soil-washing tech- nique. Four washed soil particles (~ 400/zm diameter) per Petri dish were plated in 2% malt extract agar. A total of 100 particles (25 Petri dishes) for each treatment were axenically incubated at 28°C during 1 week. The growing fungi species were identified and the relative colonization frequency was calculated according to the following (Godeas, 1983): %frequency = colonized particles x 100/total particles Chemical analyses Aliquots of about 0.5 g of carefully homogenized soil samples were subjected to dichloromethane-ultrasonic extraction (five times, 10 min each). After centrifugation, the whole extract was evaporated under a stream of nitrogen to constant weight to determine the total dichloromethane extract (TDCL). Separation of the aliphatic (ALI) and aromatic (ARO) fractions was achieved by microcolumn chromatography using Pasteur pipettes filled with 3% deactivated silica gel and anhydrous NaSO4. The elution volumes (3.5 ml petro- leum ether for ALI and 4 ml of a 3:1 petroleum-ether- dichloromethane mixture for ARO) were empirically adjusted with ALI (Cl 5-30 n-alkanes) and ARO (fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrisene, benzo[b]fluoranthene, benzo[k]fluoranthene) authentic standards (Polyscience Corp.). Aliphatic and aromatic hydrocarbons 357 C D 3-2 9-1 MPhe MPhel A~.._.ad.~ y ,'0 2'0 3'0 Retention time (rain) Fig. 2. Gas chromatograms of the aromatic fraction of the initial 10% oil-polluted soil (A) and the intermediate decom- position stages (60 days) of Peniciilium chrysogenum (B), Aspergillus terreus (C) and Fusarium solani (D) treatments. Phe, 3-2-MPhe and 9-l-MPhe stand for phenanthrene, 3-2-methyi- phenanthrenes and 9-l-methylphenanthrenes. The arrows mark some non-quantified peaks with contrasting behaviour: rapidly lost early eluting peaks (alkylated naphthalenes) and more persistent peaks after 9-1-MPhe (dimethylphenanthrenes). 120 110 100 90 80 ~ 70 × 60 N 50 ~ 40 "N [-, 20 10 0 2 0 n8~ 16 ~- ,E~ 14~- ,2~ .~ io ~- ._~ 8 - 6~ ~- 4 ~ 2~ 0: 152 141 A l i p h a t i c H C / a r o m a t i c H C 56 H , ¢ / x / / ~ / / / z / x ~ / / / / / / / / / / / z J ~ Initial Natural Peni 386 135 88 - - - - / / A / / / f/A ~"A / / A 9"A VA ' / A / / ] Fusa Asper 154 109 153 - - - - ,-,.,. ~ / / / / / I ~ / / / / / / i / / / f ] r f ~ z / / f i / r / / / / / i / t Pleuro Corin Tricho Trame 1.85 Initial Natural Fusa Peni n -C 16+n-C 17+n-C 1 8 / N p r + P r + P h y t 0.99 1.18 1.20 Pleuro Corio Asper Tricho Trame Quantification of individual hydrocarbons was carried out by high-resolution gas chromatography (GC) using a Shimadzu GC-7AG gas chromatograph equipped with a 0.25 mm×30 m SPB-5 silica column, a split-splitless injector (split ratio=8) and a flame ionization detector operated at 320°C. The column temperature was programmed from 135 (1-min hold) to 295°C (8-min hold) at 3.5°C/min. Hydrocarbons were quantified using individual response factors obtained from the pure standards mixtures that were injected at least once during each working day. The ALI compo- nents quantified included C15-C30 n-alkanes and three major isoprenoids (norpristane, pristane and phytane; Fig. 1). In the ARO fraction, only the very abundant and well-characterized phenanthrene, 3-2- and 9-1-methylphenanthrenes (MPhe) were quantified. The assignment of 3-2- and 9-l-MPhe was accomplished by comparison with published chromatograms and the calculation of retention indexes as previously reported for the characterization of contaminated local river sediments (Colombo et al., 1989, and references therein). Due to the lack of a more definitive identity confirmation, other relatively abundant peaks, eluting close to the solvent (probably alkylated naphthalenes) and after methylphenanthrenes (i.e. dimethylphen- anthrenes), were not quantified (Fig. 2). Typically, the individual response factors derived from the standards varied by + 10% and the reproducibility of the analyses tested with duplicate samples averaged + 15%. 0.14 0.12 0.10 "v 0.08 E 0.06 "~ 0.04 0.02 0 3 - 2 M P h e n a n t h r e n e / 9 - 1 M P h e n a n t h r e n e 0.90 ~/ 0.96 ~ -- 0.94 Initial Natural Peni 0.64 0.53 0.67 Fusa Pleuro Corio Asper Tricho Trame Fig. 3. Average concentrations of the total dichloromethane extract (TDCL) and total CG-resolved aliphatic and aromatic hydrocarbons in the initial 10% oil-polluted soil and the eight treatments. The average aliphatic/aromatic, n-alkane/isoprenoid and 3-2-methylphenanthrene/9-l-methylphenanthrene ratios are shown above the bars. RESULTS AND DISCUSSION Soil colonization After 90 days of incubation, the fungi colonization efficiency ranged from a minimum of 50% for the natural soil microflora to 100% for the particles for Hypho- mycetes. The individual treatment results were as follows: Penicillium chrysogenum, 60%; indigenous soil microflora, 358 J .C . Colombo et al. Table 1. Concentrations (mg]g dry wt) on days 0, 15, 30, 60 and 90 of aliphatic and aromatic hydrocarbons and total dichioromethane extract in the initial 10% oil-contaminated soil and the eight biodegradation treatments 10%soil Penicilliumchrysogenum Naturalsoilmicroflora Aspergillus t erreus Fusarmmsolani 0 15 30 60 90 15 30 60 90 15 30 60 90 15 30 60 90 days days days days days days days days days days days days days days days days days Aliphatic hydrocarbons n-C15 - - 0.37 0.15 0.15 0.00 0.76 0.34 0.30 0.00 0.33 0.00 0.06 0.17 0.39 0.21 0.38 0.30 n-C16 1.45 0.43 0.20 0.33 0.28 0.86 0.48 0.45 0.59 0.35 0.19 0.13 0.17 0.45 0.32 0.42 0.36 Norpr 0.67 0.23 0.15 0.23 0.25 0.41 0.21 0.28 0.59 0.20 0.13 0.15 0.20 0.22 0.22 0.27 0.27 n-C17 1.71 0.42 0.29 0.35 0.29 0.92 0.54 0.61 0.96 0.36 0.23 0.23 0.20 0.51 0.39 0.49 0.43 Pris 0.89 0.31 0.28 0.34 0.36 0.62 0.35 0.42 0.75 0.21 0.21 0.29 0.35 0.38 0.46 0.41 0.40 n-Ci8 1.66 0.41 0.26 0.30 0.24 0.85 0.53 0.56 0.93 0.30 0.23 0.21 0.19 0.47 0.41 0.46 0.38 Phyt 1.06 0.32 0.28 0.35 0.36 0.55 0.33 0.40 0.74 0.23 0.21 0.28 0.33 0.35 0.38 0.39 0.41 n-Cl9 1.50 0.33 0.29 0.27 0.24 0.72 0.50 0.66 0.96 0.26 0.24 0.23 0.19 0.40 0.44 0.42 0.34 n-C20 1.42 0.35 0.30 0.24 0.20 0.61 0.46 0.72 0.76 0.23 0.22 0.24 0.15 0.38 0.41 0.38 0.32 n-C21 1.13 0.28 0.23 0.21 0.19 0.49 0.35 0.56 0.74 0.20 0.19 0.20 0.12 0.32 0.29 0.30 0.27 n-C22 0.95 0.25 0.23 0.19 0.16 0.41 0.31 0.52 0.56 0.18 0.17 0.20 0.14 0.28 0.27 0.26 0.24 n-C23 0.80 0.20 0.20 0.17 0.16 0.35 0.25 0.46 0.52 0.13 0.14 0.16 0.09 0.23 0.24 0.22 0.16 n-C24 0.76 0.17 0.19 0.15 0.13 0.31 0.23 0.41 0.44 0.10 0.13 0.15 0.09 0.19 0.22 0.21 0.16 n-C25 0.67 0.15 0.15 0.13 0.10 0.24 0.19 0.34 0.40 0.09 0.12 0.14 0.08 0.16 0.18 0.18 0.13 n-C26 0.54 0.11 0.13 0.09 0.09 0.17 0.17 0.29 0.29 0.07 0.10 0.11 0.06 0.11 0.15 0.15 0.09 n-C27 0.42 0.11 0.12 0.08 0.08 0.16 0.12 0.27 0.27 0.05 0.08 0.08 0.06 0.10 0.12 0.12 0.10 n-C28 0.31 0.06 0.11 0 .07 0.08 0.09 0.10 0.21 0.27 0.04 0.06 0.07 0.04 0.11 0.11 0.10 0.07 n-C29 0.34 0.07 0.06 0.07 0.07 0.10 0.08 0.24 0.25 0.03 0.06 0.05 0.04 0.07 0.11 0.09 0.08 n-C30 0.24 0.07 0.06 0.06 0.07 0.09 0.06 0.22 0.18 0.03 0.06 0.10 0.04 0.06 0.10 0.09 0.06 Total ALl 16.5 4.6 3.7 3.9 3.4 8.7 5.6 7.9 10.2 3.4 2.8 3.1 2.7 5.2 5.0 5.4 4.6 Aromatic hydrocarbons Phe 0.03 0.02 0.02 0.02 0.02 0.01 0.02 0.02 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3-2-MPhe 0.04 0.02 0.02 0.03 0.03 0.02 0.02 0.02 0.02 0.02 0.02 0.01 0.00 0.01 0.00 0.00 0.00 9-1-MPhe 0.04 0.02 0.03 0.03 0.03 0.02 0.02 0.03 0.02 0.02 0.03 0.01 0.00 0.02 0.01 0.01 0.00 Total ARO 0.11 0.06 0.07 0.08 0.07 0.05 0.05 0.07 0.05 0.06 0.05 0.03 0.00 0.03 0.01 0.01 0.00 Total DCL 91.3 56.3 72.7 72.9 69.0 76.9 91.4 91.6 87.0 50.3 67;3 58.5 60.0 51.8 63.7 73.3 67.7 Ratios n-CiT/Pris 1.92 1.37 1.07 1.04 0.80 1.48 1.55 1.45 1.31 1.71 1.14 0.79 0.57 1.34 0.84 1.19 1.07 n-Ci8/Phyt 1.57 1.27 0.96 0.86 0.67 1.54 1.62 1.37 1.26 1.33 1.11 0.77 0.57 1.35 1.09 1.20 0.91 n-Ci6_ls/Isopr 1.85 1.47 1.07 1.07 0.83 1.66 1.75 1.45 1.20 1.60 1.22 0.80 0.63 1.50 1.06 1.26 1.08 Phe/MPhe 0.37 0.39 0.34 0.30 0.36 0.38 0.50 0.35 0.36 0.19 0.04 0.04 0.00 0.14 0.10 0.00 0.00 3-2/9-l-MPhe 0.90 1.10 0.85 0 .77 1.11 0 .89 1.06 0.68 1.12 1.13 0.86 0.80 0.50 0.88 0.44 0.40 0.00 continued opposite 50%, composed of Fusarium oxysporum (20%), Talaro- myces rotundus (10%), Peni¢illium sp. (5%), Trichoderma koningii (5%), Mucor hiemalis (3%), Alternaria alternata (2%), Cladosporium cladosporioides (2%), Penicillium thomii (2%) and Absidia sp. (1%); Aspergillus terreus, 100%; Fusarium solani, 100%; Trichoderma harzianum, 100%; Pleurotus ostreatus, 80%; Trametes villosa, 85%; and Coriolopsis rigida, 75%. The low colonization efficiency of the natural soil microflora indicate that these unadapted microorganisms, which include the previously enumerated fungi species plus bacteria (not studied in this work), are unable to successfully grow in 90 days in an oil-polluted media. This explains the poor degradation performance observed for this treatment (see next section). Overall oil degrading performances An intercomparison of the global degradation capabilities of the soil microflora and fungi is presented in Fig. 3, which shows the 15-90 day average con- centrations of the total dichloromethane extract (TDCL) and total GC-resolved ALl and ARO hydro- carbons in the initial 10% oil-polluted soil and the eight treatments. Table 1 presents the complete data set for all the treatments. The TDCL histograms show that, excepting the natural soil microbial community, whose TDCL levels (87+6.9 mg/g) overlap with the initial polluted soil values (91 + 15 mg/g), all fungi species sig- nificantly reduced TDCL concentrations to 59-68 mg/g (individual values range from 50 to 75 mg/g; Table 1). Some subtle differences in efficiency are insinuated (higher reduction for A. terreus and lower for P. chrysogenum), but for these 15-90 day-average TDCL values the differences are not significant (ANOVA; p=0.05). An indication of the ALl versus ARO selectivities is also shown. Overall, aliphatic hydrocarbons seem to be more easily degraded than AROs. However, there are some interesting distinctions. A strong preferential ALl decay is clearly indicated for A. terreus and specially P. chrysogenum (low ALI /ARO values) whereas F. solani shows a proportionally higher reduction of ARO (highest ALI/ARO). T. harzianum and C. rigida also seem to favour ALl decomposition, whereas P. ostreatus and T. Aliphatic and aromatic hydrocarbons Table 1--Continued 359 Trichoderma harzianum 15 30 60 90 days days days days P&urotus ostreatus Trametes villosa Coriolops~ rigida 15 30 60 90 15 30 60 90 15 30 60 90 days days days days days days days days days days days days Aliphatic hydrocarbons n-C15 0.60 0 .00 0.11 0 .00 0.50 0.00 0 .15 0 .19 0 .46 0 .25 0.11 0.25 - - 0 . 3 0 0 .13 0.17 n-Ct6 0.68 0 .52 0 .22 0 .19 0 .56 0 .38 0 .20 0 .22 0 .52 0 .30 0 .17 0 .25 0 .54 0 .37 0 .20 0.20 Norpr 0.36 0.36 0.21 0 .23 0 .26 0 .22 0 .18 0.21 0 .26 0 .17 0.14 0 .22 0 .26 0 .20 0 .17 0.19 n-Ci7 0.81 0 .63 0 .29 0 .23 0 .62 0 .45 0 .26 0.26 0 .57 0 .35 0.24 0.26 0 .64 0 .43 0 .25 0.24 Pris 0.63 0 .67 0.44 0 .37 0 .42 0 .40 0.31 0.31 0 .40 0 .27 0 .29 0 .34 0 .43 0 .49 0 .38 0.26 n-Cls 0.78 0 .70 0.36 0 .24 0.56 0 .47 0 .29 0 .24 0 .50 0.31 0 .28 0 .30 0 .63 0 .47 0 .29 0.23 Phyt 0.62 0 .63 0 .45 0 .39 0.40 0 .36 0 .27 0 .33 0 .37 0 .27 0 .26 0 .35 0 .45 0 .37 0.31 0.26 n-Ci9 0.74 0.74 0 .35 0 .22 0 .56 0 .47 0 .35 0 .22 0 .37 0 .27 0 .32 0 .23 0 .57 0 .52 0 .37 0.22 n-C20 0.72 0.70 0 .42 0.20 0.44 0 .39 0 .38 0 .18 0 .37 0.24 0 .32 0.21 0 .47 0.54 0 .37 0.19 n-C21 0.50 0 .64 0 .27 0.16 0 .36 0 .32 0 .30 0 .14 0 .30 0.20 0 .22 0 .15 0 .39 0 .45 0.26 0.15 n-C22 0.46 0 .52 0 .26 0 .15 0 .25 0 .29 0 .27 0 .14 0 .25 0 .19 0 .20 0 .14 0 .35 0 .43 0 .25 0.12 n-C23 0.41 0 .46 0 .24 0 .13 0.24 0.26 0 .23 0.11 0.21 0 .16 0 .18 0 .12 0 .30 0.36 0 .22 0.10 H-C24 0.37 0 .45 0 .23 0.11 0 .23 0.24 0.21 0 .10 0 .19 0 .14 0 .17 0 .10 0 .27 0 .35 0 .22 0.10 n-C2~ 0.31 0 .39 0 .20 0.11 0 .19 0 .20 0 .18 0 .09 0 .17 0.11 0 .16 0 .08 0 .19 0.31 0.21 0.09 H-C26 0.27 0 .35 0 .16 0 .09 0 .15 0 .16 0.14 0 .08 0 .13 0 .10 0.11 0.06 0 .16 0 .26 0.14 0.06 H-C27 0.21 0 .27 0 .14 0 .06 0 .10 0 .13 0.11 0 .06 0 .09 0 .07 0.10 0 .05 0 .15 0.21 0.11 0.05 n-C28 0.18 0 .22 0 .14 0 .05 0 .09 0.11 0 .09 0.04 0 .09 0 .06 0 .10 0 .04 0.11 0 .17 0 .10 0.04 n-C2~ 0.16 0 .23 0 .15 0.04 0 .10 0 .10 0 .10 0 .05 0 .10 0 .05 0 .09 0 .05 0 .10 0 .15 0 .13 0.04 n-C3o 0.17 0 .19 0 .15 0 .05 0 .08 0 .10 0 .10 0 .04 0 .06 0 .05 0 .10 0 .05 0 .06 0 .14 0 .13 0.04 Total ALl 9.0 8.7 4.8 3.0 6.1 5.1 4.1 3.0 5.4 3.6 3.6 3.3 5.9 6.5 4.3 2.8 Aromatic hydrocarbons Phe 0.02 0.01 0 .00 0.00 0.01 0 .00 0 .00 0 .00 0.01 0 .00 0 .00 0 .00 0.01 0 .00 0 .00 0.00 3-2-MPhe 0.03 0 .03 0.01 0 .00 0 .03 0.01 0.01 0 .00 0 .02 0 .02 0 .02 0 .00 0.01 0 .02 0 .00 0.00 9-1-MPhe 0.04 0 .03 0 .02 0 .00 0.04 0 .02 0.01 0 .00 0 .02 0 .02 0 .03 0 .00 0 .02 0 .02 0 .00 0.01 Total ARO 0.09 0 .07 0 .03 0.00 0 .07 0 .03 0.01 0 .00 0 .05 0 .04 0 .05 0 .00 0.04 0.04 0.01 0.01 Total DCL 73.4 65 .5 55 .4 57.6 69 .6 58.8? 61.0 61 .6 73.1 65 .8 65 .0 56 .9 52.8 59.3 65 .0 74.9 Ratios n-Cw/Pris 1.29 0 .93 0.64 0.61 1 .46 1.12 0.81 0 .84 1.43 1.30 0.84 0 .83 1.48 0 .88 0.66 0.86 n-Cis/Phyt 1.26 1.12 0 .80 0 .63 1 .39 1.24 1.06 0 .73 1.34 1.16 1.00 0 .86 1.40 1.28 0 .96 0.82 n-C,, 18/lsopr 1.42 1.11 0 .78 0 .67 1.60 1.30 0 .96 0.84 1.54 1.36 0 .99 0 .92 1.59 1.20 0 .88 0.89 Phe/MPhe 0.26 0 .16 0 .07 0 .00 0 .13 0 .03 0 .00 0 .00 0.21 0 .07 0 .00 0.00 0.21 0 .04 0 .00 0.00 3-2/9-I-MPhe 0.87 1.07 0.61 0 .00 0 .83 0 .65 0 .66 0 .00 0 .95 0 .97 0 .77 0 .00 0 .73 0 .75 0 .34 0.00 Total ALl, total resolved aliphatic hydrocarbons; Total ARO, total aromatic hydrocarbons; TDCL, total dichloromethane extract; Norpr. Norpristane; Pris, pristane; Phyt, phytane; Isopr, norpristane + pristane + phytane; Phe, phenanthrene; MPhe, methylphenanthrenes. --- not determined. villosa do not appear to have any preference (ALI /ARO to the initial polluted soil: 152). To estimate the possible contribution of abiotic losses (namely volatilization) to the total decay, the decrease of TDCL levels and the degree of ALI alteration relative to the initial polluted soil was evaluated for the least active treatment, the natural soil microflora, which showed the highest TDCL levels and the lower alteration. The average decrease of TDCL in this treatment is 5% (91-87 mg/g) or a maximum of 13% if the theoretical 100 mg/g oil addition is considered. However, not all this decrease is abiotic loss because the ALI suite shows signs of partial decay (alkane/isoprenoid ratios = 1.5 versus 1.8 in the original crude). Thus, an average volatilization loss of about 10% seems a reasonable upper estimate, specially considering that the other fungi species have much higher degradation capabilities and that the 1-kg soil samples were kept in closed 4-1itre glass jars throughout the experiment. For total GC-resolved ALIs, all eight treatments, including the natural soil community, showed a marked reduction of hydrocarbon levels. However, the differences in degradation efficiency are much more pronounced than for TDCL. Both the ALI concentrations and the alkane/isoprenoid ratios (Fig. 3) indicate that, relative to the initial polluted soil ( A L I = 16 mg/g; alkane/isoprenoid = 1.8), A. terreus is the more efficient degrading species ( A L I = 3 mg/g; alkane/isoprenoid = 1.1), whereas the natural soil assemblage presents the lowest capacity ( A L I = 8 rag/g; alkane/isoprenoid -- 1.5). The other fungi species present intermediate efficiencies. A somewhat different degradation efficiency trend is indicated for the ARO fraction. In this case, the alteration index is the phenanthrene/methylphenanthrenes ratio (Phe/MPhe) or the 3-2/9-t-MPhe ratio, which permits the evaluation of more advanced degradation stages, since phenanthrene is readily degraded (Fig. 2) and the Phe/MPhe ratio becomes rapidly zero. Again, as for the aliphatic fraction, both the ARO concentrations and the alteration ratios relative to the initial polluted soil (ARO=0.11 mg/g; Phe/MPhe=0.37; 2-3/9-1-MPhe = 0.90) indicate a consistent picture: F. solani is the more efficient degrading species (ARO = 0.03 mg/g; Phe/MPhe = 0.06; 3-2/9-l-MPhe = 0.43), whereas P. cho,sogenum 360 J .C . Colombo et al. and the natural microbial community show the highest concentrations and ratios (ARO=0.06--0.07 mg/g; Phe/MPhe=0.35-0.40; 3-2/9-1-MPhe=0.94--0.96) indi- cating little degradation capacity. The other species show intermediate performances. These results indicate that the natural microbial soil assemblage isolated from an urban forest area with no history of heavy oil pollution was incapable of significantly degrading crude oil, whereas pure fungi cultures effectively reduced the residues. The total oil residue (TDCL) loss due to biodegradation ranged from 26 to 35%. These values probably represent an under- estimation of maximum degrading potentials due to the limiting (more extensive) experimental conditions (high hydrocarbon load, no fertilization and unadapted Basidiomycetes). Longer and more intensive treatments performed with lower loads of lighter hydrocarbons yielded higher losses: 75% reduction in 270 days in fuel- oil-contaminated (2.7 mg/g) soil microcosms (Cha~neau et al., 1995) or 83% reduction in 90 days for intensively bioremediated soils contaminated with 60 mg/g of diesel oil (Wang et aL, 1990). Aspergillus terreus and Fusarium solani, isolated from oil polluted areas, produced the strongest decay showing a selectively more efficient attack of ALIs and AROs, respectively. Overall, imper- fect fungi isolated from polluted areas showed a some- what higher efficiency, but the performance of lignolitic species was very good, especially considering that these are indigenous species isolated from unpolluted sites which were forced to live in a media with a low abun- dance of their specific substrate (cellulose and lignin). Dynamics of hydrocarbon biodegradation Figure 4 presents the evolution of the percentage of total loss of ALI and ARO hydrocarbons quantified during the 90-day experience. ALI hydrocarbons were readily degraded and the most important reduction occurs rapidly during the first 15 days (35-70%). Only a slight reduction or almost constant values are observed for the rest of the experience. T. harzianum was the sole species showing a continuous and progressive degrading activity. The natural soil microflora oscillated around a lower 50% total decrease. A different dynamics is observed for the ARO frac- tion. Except perhaps F. solani, the most effective decomposer, which almost completely depleted phenan- threne and methylphenanthrenes in the first 30 days, the other species showed a slower kinetics. Maximum degradation efficiencies were attained only at day 90, reflecting the well known more recalcitrant character of ARO hydrocarbons relative to ALIs (Atlas, 1981; Fusey & Oudot, 1984; Cha~neau et al., 1995). The less efficient natural soil assemblage and P. chrysogenum fluctuated around a 40% total decrease all over the experience. As occurred with the ALI fraction, T. harzianum also shows a gradually increasing ARO degradation activity. Both observations taken together suggest that T. har- zianum has a slower enzymatic kinetics or that it needs a longer activation period in the presence of oil residues. Nevertheless, by the end of the experience the degrading 100 ~ 8 0 _o 60 .. ~ 4o ~ 2o V o l a t i l i z a t i o n 15 30 415 610 75 90 I i VVolatilization 0' 15 3~) 45 6~) 75 9{) Time (days) • Peni o Natural • Asper x Fusa * Tricho x Pleuro o Trame zx Corio Fig. 4. Evolution of the percentage of total loss of the aliphatic and aromatic hydrocarbons quantified during the 90-day experience. performance of T. harzianum for both the ALl and the ARO fractions is as good as those of the other species. Reactivities of hydrocarbons during biodegradation The progress of degradation resulted in marked com- positional changes in the oil residue (Figs 1 and 2). As expected, the most conspicuous trend is the rapid relative decrease of lower-molecular-weight (LMW) n-alkanes (n-Ci5 to n-C18) and the increase of isoprenoids. There is also an intermediate (days 30-60) increase in the proportion of higher-molecular-weight (HMW) n-alkanes. In the ARO fraction, there is a rapid loss of phenanthrene and relative enrichment of 9-1-methylphenanthrenes along the experience. A similar change is insinuated for some of the non-quantified compounds: early eluting peaks (i.e. alkylated naph- thalenes) are rapidly lost, whereas the major peaks after 9-l-methylnaphthalenes (i.e. dimethylnaphthalenes) are more persistent (Fig. 2). To evaluate the covariation of the many different parameters and the grouping of samples (treatments) along the experience, a principal component analysis was performed with the standardized relative abun- dances of 22 hydrocarbons (norpristane, pristane, phytane, C15-30 n-alkanes, phenanthrene, 3-2- and 9-1-MPhe). Figure 5 shows the correlation of the original variables (close rays = positive, opposite rays = negative) and the grouping of samples (symbols) represented together in the plane of the first two principal components to facilitate the interpretation of the results. Aliphatic and aromatic hydrocarbons 361 2 ~ o o -2 -4 O ,.c30 / / / ' Norpr • "r-' \W,, 3,2-MPhe I \""~\~::j n -C 16 Phe n-CI8 n-CI7 [] % A ° I I I I I "66 -4 -2 0 2 4 6 Component I Form reference • Tricho 41= Pleuro • Natural • Corio • Trame "k Peni • Fusa O Asper [ ] Initial Fill reference [ ]Day 15 [ ] D a y 3 0 • D a y 6 0 • D a y 9 0 Fig. 5. Simultaneous representation of the original variables (rays; close rays = positive correlation, opposite rays = negative correlation) and samples (symbols) in the plane of the first two principal components (PCI =50% and PC2=24% total variability). The principal component analysis was carried out with the standardized relative abundance of 22 hydrocarbons. These results indicate a clear reactivity trend of the components during oil biodegradation: LMW n-alkanes > phenanthrene >3-2-MPhe > intermediate HMW n-alkanes > longer chain H M W n-alkanes > isoprenoids ,~9-1-MPhe. Irrespective of their degrading capacity, all fungi species tested seemed to pass through this decomposition sequence. For a fixed time, their position along the sequence would be determined by their performance. A. terreus and Trichoderma harzianum arrive at a more degraded stage of ALl alteration in 90 days. F. solani readily degrades phenanthrene and 3-2-MPhe arriving faster at the stage of 9-1-MPhe pre- dominance, whereas the natural soil assemblage does not arrive at the stage of relative enrichment in iso- prenoids. This reactivity trend is very similar to that reported by Hostettler & Kvenvolden (1994) for 17-38- month weathered residues of the oil spilled from the T/V Exxon Valdez. ALIs disappeared in the order LMW n-alkanes, H M W n-alkanes and isoprenoids, whereas in the ARO fraction, the sequence was also phenanthrene, 3-2-MPhe and 9-1-MPhe. ACKNOWLEDGEMENTS The authors are indebted to M. Dabadie and C. Picone for their technical assistance. This paper has benefited from the constructive criticism of two anonymous reviewers. This research was supported by Grants from CONICET and CIC, Argentina. REFERENCES The amount of the total variability accounting for the first and second principal components is 50 and 24% respectively. Principal component 1 (PC1) has a strong contribution of the group of LMW n-alkanes (n-Cts-18, r=0.13-0.90; +PC1) and HMW n-alkanes (n-C20-30, r=0.434).94; - P C I ) which are negatively correlated (n-C15 18 versus n-C26-30, r = -0.40 to -0.83). 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