Isolation of autochthonous non-white rot fungi with potential for enzymatic upgrading of Venezuelan extra-heavy crude oil

The increasing world demand for fuels makes it necessary to exploit the largest reserve of extra-heavy crude oil (EHCO) of the Orinoco Oil Belt from Venezuela. We propose the use of extracellular oxidative enzymes, in particular, lignin-degrading enzyme systems (LDS) of fungi, for enzymatic improvement of EHCO. Autochthonous non-white rot fungal strains able to use EHCO, and several polycyclic aromatic hydrocarbons (PAHs) as sole carbon source and energy, were isolated from EHCO-polluted soils and identified as belonging to the genera Fusarium, Penicillium , Trichoderma , Aspergillus , Neosartorya, Pseudallescheria, Cladosporium, Pestalotiopsis , Phoma and Paecillomyces. Phenotypic and biochemical assays revealed the ability of these filamentous fungi to synthesize extracellular oxidative enzymes, and suggested a relationship between the LDS and EHCO bioconversion. This work reports, for the first time, the use of o-phenylenediamine dihydrochloride (OPD) as substrate to measure extracellular ligninolytic peroxidases (ELP) in culture broths of filamentous fungi (Fusarium solani HP-1), and constitutes the first formal study of the fungal community associated with the EHCO of the Orinoco Oil Belt.


Introduction
The complementary use of biotechnology in the petroleum industry provides new tools to improve heavy and extra-heavy crude oil (EHCO). Increasing world demand for fuels (estimated at 520 million barrels of petroleum for 2007) makes it necessary to exploit the extensive deposits of the Orinoco Oil Belt from Venezuela, the biggest reservoir of EHCO in the world, estimated at 1.5 trillion barrels (PDVSA 2002). Besides being particularly rich in heteroatoms (nitrogen, sulfur and oxygen), and metals such as nickel and vanadium, the EHCO of the Orinoco Oil Belt contains a high concentration of resins and asphaltenes, the two most polar fractions and with the highest molecular weight of EHCO. The latter are responsible for EHCO's high viscosity, which makes it difficult to extract, transport and refine by conventional methods (León 1998; for reviews, see Leó n & Kumar 2005). This work focuses on the development of the enzymatic improvement of these Venezuelan non-conventional hydrocarbons using the powerful extracellular oxidative lignin-degrading enzyme system (LDS) of fungi as biological catalysts.
Enzymatic processes may provide an alternative to conventional methods for EHCO upgrading, having the advantage of being environmentally friendly and economically sound. LDS could change the physical properties of EHCO without the loss of carbon atoms, and keeping the calorific value of the hydrocarbons. Partial enzymatic oxidation of the internal aliphatic linkage (sulfides, esters, and ethers) and of PAHs, resins and asphaltene molecules can lead to a reduction in total aromaticity of EHCO, decreasing its viscosity and density levels (Kirkwood et al. 2004). Thus, the great interest in using fungal extracellular oxidative enzymes from LDS for enzymatic EHCO upgrading. If non-white rot fungal strains contain the LDS and it is involved in EHCO bioconversion, then EHCO addition as sole carbon source to culture broth should induce the extracellular oxidative activities from LDS. Filamentous fungi with the ability to produce extracellular oxidative enzymes on culture plates were identified by ABTS-oxidizing activity, as described previously by Saparrat et al. (2000), whereas the detection of extracellular ligninolytic peroxidase (ELP) activities in culture broths of filamentous fungi was performed using a rapid and efficient method, the o-phenylenediamine dihydrochloride (OPD) test. OPD is a very stable chromogen, water-soluble and a sensitive substrate used for diagnostic applications and immunochemical technique to measure peroxidase activity (Wolters et al. 1976;Bovaird et al. 1982). Interestingly, preliminary assays revealed that some autochthonous non-white rot fungi associated with EHCO are able to synthesize extracellular oxidative enzymes from LDS; this, in turn, suggests that these enzymes are implicated in EHCO bioconversions.
The principal aim of this study was to isolate, identify, and characterize autochthonous fungal strains with high potential to metabolize PAHs and EHCO. These filamentous fungal strains will be used as potential biocatalytic agents, to design novel green processes aimed at exploiting and rationally using natural resources, such as the Orinoco Oil Belt, and guarantee sustainable development of Venezuela.

Isolation of autochthonous fungus and culture conditions
EHCO-polluted soils samples (ca. 5 g) from Sucre and Miranda states (Venezuela) were tested. Initially, the strains isolated were screened by selecting for their abilities to grow on Czapek (Cz) minimal medium (Peláez et al. 1995;Naranjo et al. 2005) supplemented with 2% (w/v) of Carabobo (formerly Cerro Negro) EHCO emulsion as sole carbon source (Cz P ). Spores from fungal strains were grown on Cz P for 7 days at 308C.

Phenotype assays from autochthonous fungal strains
Autochthonous fungal strains with the ability to grow on Cz P were tested for their ability to grow on Cz supplemented with several PAHs (naphthalene, dibenzothiophene, phenanthrene, or pyrene) as sole carbon source and energy at a final concentration of 200 ppm. The plates were incubated for 7 days at 308C in the dark, and growth of the fungal strains was analyzed. A scale based on colony growth was used for interpreting the results: non-growth ((); residual growth ('/ (); weak growth ('); moderate growth (''); and good growth (''').

Isolation of fungal genomic DNA
Spores from fungal strains were inoculated into MPPY medium (Montenegro et al. 1992;Naranjo et al. 2001Naranjo et al. , 2004 and incubated in an orbital shaker at 250 rpm for 36 Á48 h at 308C. The resulting mycelium was recovered, filtered through Nytal filters, washed twice with 0.9% (w/v) NaCl, frozen with liquid nitrogen and stored at (808C. Mycelium samples (ca. 500 mg) were treated with 1 mL of 0.18 M Tris/HCl pH 8.2; 10 mM EDTA; 1% SDS and 1 mL of phenol, and were incubated for 30 min at 508C. Then, phenol ÁCIA treatment (phenol: chloroform:isoamyl alcohol, 25:24:1) was repeated until the interface was clear. Genomic DNA was precipitated with 2.5 vol of ethanol and 0.1 vol of 3 M sodium acetate (pH 3.2) and resuspended in Tris ÁEDTA (TE) buffer (Sambrook et al. 1989).

Classic and molecular identification of autochthonous fungal strains
Fungal strains were classified by macro and microscopic studies of morphological characters (hyphae, conidia, chlamydospores, conidiogenus cells and conidiophores). The information was compiled in a taxonomic description for comparison with specialized literature. For molecular identification of autochthonous EHCO-degrading fungal strains, PCR amplifications of 28S rRNA gene were performed using the following specific primers: NL1 (forward): 5?-GCATATCAATAAGCGGAGGAAA AG-3? and NL4 (reverse): 5?-GGTCCGTGTTTC AAGACGG-3?. PCR reactions were performed in an Applied Biosystems 2720 thermocycler using a final concentration of 0.25 mM of each deoxyribonucleotide triphosphate, and 100 Á300 ng of fungal genomic DNA as template. PCR conditions consisted of an initial denaturation at 958C for 5 min, 40 cycles of amplification at 958C for 35 s, annealing at 528C for 30 s, extension at 728C for 20 s, and final extension at 728C for 10 min. The PCR product (600 bp) was doubly purified using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI) and sequenced using BigDye TM Terminator v.3.1 kit (Applied Biosystems) on an ABI Prism TM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Several DNA samples were sent to the UC Berkeley DNA Sequencing Facility (California, USA). All other nucleic acid manipulations were carried out by standard methods (Sambrook et al. 1989).

Detection of extracellular oxidative enzymes on culture plates
The ability of the fungal strains to produce extracellular oxidoreductases of LDS was performed by the 2,2?-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS; Sigma-Aldrich) test as described previously by Saparrat et al. (2000). The chromogen ABTS is a very sensitive substrate that allows rapid screening of fungal strains producing the extracellular oxidative enzymes by means of a colorimetric assay at 420 nm (Saparrat et al. 2000). Each strain was processed in duplicate under controlled conditions at 308C in darkness, for 8, 15 and 21 days. A scale based on the color intensity was used for interpreting the results: colorless (() indicates no ABTS-oxidizing activity; low color intensity ('); moderate color intensity (''); and high color intensity (''') indicating high ABTS-oxidizing activity.

Detection of extracellular ligninolytic peroxidase activities in culture broths
Fresh spores were inoculated into MPPY medium and incubated in an orbital shaker at 250 rpm and 308C. The mycelium grown-up was used as the inoculum for cultures in different conditions: (i) Cx medium (Cz medium with N-limited conditions); (ii) Cx supplemented with naphthalene as sole carbon source and energy; and (iii) Cx with 1% (w/v) of Carabobo-EHCO emulsion as sole carbon source and energy. Cultures were incubated at 308C, and the supernatants collected at 48, 72, 96, 120, and 168 h post-inoculation and used for enzymatic assays. The detection of ELP activities was per-formed using the OPD oxidation test according to the manufacturer's instructions (Pierce; Rockford, USA) with some modifications. The reaction mixture contained 0.1 M citric/sodium phosphate buffer at pH 6.8; 0.75 mg mL (1 OPD; 20 mL of hydrogen peroxide 30% and 100 mL of sample (mycelium and EHCO-rest supernatant free) in a final volume of 1 mL. The mixture was incubated at room temperature for 30 min. OPD oxidation yields a soluble end product which is yellow Áorange in color, and was monitored over 30 min at 490 nm using a Perkin-Elmer Instruments Lambda 35 UV/VIS spectrophotometer running UVwinlab version 2.85.04 2000 software. All reactions were carried out in duplicate, at least. The extinction coefficient used was 1.578 mM (1 cm (1 , as determined previously by Romano et al. (2000). One OPD unit is defined as the amount of enzyme that oxidizes 0.01 mmol OPD per min (Romano et al. 2000). Protein concentration was determined by the Bradford assay (Pierce).

Results and discussion
Ability of isolated fungal strains to use EHCO and PAHs as sole carbon sources The potential of extracellular oxidative enzymes from LDS in remediation of polluted soils and oxidation of high-molecular-weight PAHs has been extensively described for white rot fungi (for a review, see Leahy & Colwell 1990;Conesa et al. 2002;Van Hamme et al. 2003a,b;Gianfreda & Rao 2004). However, very little is known about nonwhite rot fungi involved in PAH bioconversion. EHCO-polluted soils were used to isolate autochthonous fungal strains able to metabolize EHCO, yielding 45 different fungal strains, which were cultivated axenically and screened by selecting for the ability to grow on Cz P (Figure 1). These strains were then tested for their ability to grow on Cz medium supplemented with several PAHs as sole carbon source and energy. This revealed that 20 different strains metabolized EHCO and various PAHs, indicating that it was possible to isolate filamentous fungi that grow under these extreme conditions using adequate selection techniques (Table I). These strains were retained for later identification. The results obtained through phenotype assays suggested that there is an enormous genetic diversity hidden in the crude oil-polluted soil linked to the PAHs and EHCO bioconversion. The enzymatic battery potentially available in this autochthonous genetic reservoir waits to be uncovered and used further in novel green processes.

Identification of fungi isolated from crude oil-polluted soil
The 20 strains previously selected were identified through both classic and molecular techniques. Interestingly, the result showed that all strains isolated from EHCO-polluted soils were non-white rot fungi (Table I), and have been reported previously as ubiquitous soil-inhabiting saprophytes (Domsch et al. 1980). They belong to six predomi- Other strains isolated belonged to other nonpredominant genera: Cladosporium (1 sp.), Paecillomyces (1 sp.), Pestalotiopsis (1 sp.), and Phoma (1 sp.). Strains of the genera Aspergillus , Cladosporium , Fusarium , Neosartorya , Paecillomyces , Penicillium , Phoma , and Pseudallescheri, have previously been reported as culturable hydrocarbon-degrading fungi (Davis & Westlake 1979;April et al. 1998;Prenafeta-Boldú et al. 2001, 2002, 2004Luykx et al. 2003;Plaza et al. 2003;Chaillan et al. 2004;Veignie et al. 2004;Verdin et al. 2005Verdin et al. , 2006. However, this is the first report of the isolation of the filamentous fungus Pestalotiopsis palmarum from crude-polluted soil and its ability to metabolize PAHs and EHCO. The autochthonous fungi isolated (Table I)  Evidence for the production of extracellular oxidative enzymes Several extracellular oxidative fungal enzymes from LDS have been involved in the degradation of diverse recalcitrant and xenobiotics compounds (Vázquez-Duhalt et al. 1994;Saparrat et al. 2000;Alcalde et al. 2002Alcalde et al. , 2006Gianfreda & Rao 2004).
In order to detect the presence of extracellular oxidative enzymes in the isolated fungal strains, ABTS-oxidizing activity was analyzed by measuring the color intensity (oxidation of ABTS) of the agar medium. This revealed that extracellular oxidoreductases were present in 72% of the fungal strains tested (Table II). According to the scale described in Materials and methods, the filamentous fungi were grouped as: (i) species without extracellular ABTSoxidizing activity: Cladosporium sphaerospermum , Fusarium proliferatum FP-1, Neosartorya spinosa NS-1 and NS-2 and Trichoderma inhamatum (similar levels were obtained with Saccharomyces cerevisiae used as negative control); (ii) species with low ABTS-oxidizing activity: Penicillium sp. NRRL-28143 PN-1 and PN-2 and Penicillium sp. 4, Pseudallescheria angusta PA-1 and PA-2, Trichoderma viride TV-1 and TV-2; (iii) species with moderate ABTS-oxidizing activity: Aspergillus fumigatus and Aspergillus terreus ; and (iv) species with high ABTS-oxidizing activity: Fusarium proliferatum FP-2 and Fusarium solani HP-1, Paecillomyces sp. and Pestalotiopsis palmarum . This last group of non-white rot fungi showed similar levels of ABTS-oxidizing activity to that observed in the white rot fungus Pleurotus ostreatus, used as a positive control at 15 Á21 days (Table II). Previous studies have reported the genera Fusarium and Paecillomyces as hydrocarbon-degrading fungi (Plaza et al. 2003;Chaillan et al. 2004;Veignie et al. 2004;Verdin et al. 2005Verdin et al. , 2006. Fusarium , Paecillomyces and Pestalotiopsis have also been reported as lignindegrading fungi (Kapoor et al. 1978;Mishra et al. 1979;Norris 1980;Kamaya et al. 1981;Regalado et al. 1997;Kluczek-Turpeinen et al. 2003;Hao et al. 2006;Saparrat & Hammer 2006;Singh & Thakur 2006). Furthermore, before the establishment of the white rot fungus Phanerochaete chrysosporium as a model for study, F. solani was reported as the most vigorous microbial degrader of lignin among microorganisms isolated from soils and proposed as a useful model to study mechanisms of microbial transformations of lignocelluloses (Ohta et al. 1979;Norris 1980). More recent studies corroborated the lignin-degrading capabilities of Fusarium species (Regalado et al. 1997). Furthermore, several studies reported the lignin-degrading capabilities of Paecillomyces fusisporus, P. varioti , The presence of extracellular oxidoreductases was observed at 8, 15, and 21 days. Colorless ((); low color intensity ('); moderate color intensity (''); and high color intensity (''').
P. inflatus , and Paecillomyces sp. and the use of the latter for removal of color in pulp and paper mill effluents (Kapoor et al. 1978;Mishra et al. 1979;Kluczek-Turpeinen et al. 2003;Singh & Thakur 2006). Likewise, the ability of Pestalotiopsis guepinii to decolorize synthetic dyes and Pestalotiopsis sp. to produce laccase which efficiently decomposed lignocellulose have been described (Hao et al. 2006;Saparrat & Hammer 2006). Our results showed the high ligninolytic potential of the genera Pestalotiopsis, Paecillomyces and Fusarium (Table II). These experiments also represent an important step in selecting autochthonous fungal strains to be used in novel green processes development. Due to its high capacity to metabolize PAHs and EHCO and its high ligninolytic potential, strain HP-1 was selected for the following experiments.

The relationship between LDS and EHCO bioconversion
Lignin is a structurally similar polymer to the resins and asphaltenes, molecules responsible for the high viscosity and density of EHCO. For this reason, the relationship between LDS and EHCO bioconversion is of interest. The first step in lignin biodegradation is oxidation by hydrogen peroxide catalyzed by ELP. Among ELP, LIPp is able to directly oxidize non-phenolic units, MNPp and LACp oxidize preferentially phenolic units, but also act on nonphenolic units when mediators, such as ABTS, are present in the reaction mixture, whereas VEPp is able to combine the catalytic properties of LIPp and MNPp (Martínez et al. 1996;Ruiz-Dueñ as et al. 1999;Saparrat et al. 2002;Gianfreda & Rao 2004;Martínez et al. 2005). If the LDS of strain HP-1 is involved in EHCO bioconversion, then EHCO addition as sole carbon source to culture broth might induce the ELP activities. In order to test this hypothesis, ELP activities in the culture broth of strain HP-1 were measured using OPD as the substrate. HP-1 strain was grown-up as described in Materials and methods. Surprisingly, the results showed that the ELP activities were present at the same basal levels when either sucrose (control) or naphthalene was used as sole carbon and energy source ( Figure 2). However, the ELP activities were strongly induced when EHCO was used as sole carbon and energy source. Under these conditions, the ELP activities increased from 48 to 120 h, being nearly 3-, 12-and 20-fold higher at 72, 96, and 120 h, respectively, compared with a control (Figure 2). A decrease of ELP activities from 120 to 168 h was subsequently observed, but always showing significantly higher levels than the control. This substantial increase of ELP activities is probably due to the polymeric nature of EHCO. Unlike naphthalene, that represents a family of molecules constituted of just two benzene rings, EHCO is comprised of a diverse collection of aromatics and PAHs, some substituted with saturated hydrocarbons and heteroatoms, which are typical components of resins and asphaltenes, the most polar and highest molecular-weight fractions found in EHCO (Leó n & Kumar 2005). Apparently, the bioconversion of compounds, such as lignin and petroleum, requires synergy of a large diversity of ELP with broad substrate specificity. Therefore, induction of ELP activities will be directly proportional to the complexity of the substrate. These results strongly suggest that: (i) HP-1 strain is able to synthesize ELP; (ii) ELP activities are strongly induced by EHCO and could be involved in EHCO bioconversion; and (iii) a putative relationship between LDS and EHCO bioconversion exists. Interestingly, preliminary studies aimed at improving EHCO with fungal biocatalytic agents, showed a decrease in the 5008C' residue fraction with a significant increase in the distillate fraction (data not shown). The LDS could also be used in environmental biocatalysis for: (i) enzymatic bioremediation of xenobiotics, recalcitrant compounds, and petroleum-polluted soils; (ii) enzymatic recovery of sludge/oil spills; and (iii) renewable and clean energy production (Alcalde et al. 2006). Our next goal is the identification of the key genes and enzymes of the ELP family involved in the enzymatic bioconversion and improvement of Orinoco Oil Belt EHCO.