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.

Large amount of drilling waste associated with the expansion of the Orinoco Oil Belt (OOB), the biggest proven reserve of extra-heavy crude oil (EHCO) worldwide, is usually impregnated with EHCO and highly salinized water-based drilling fluids. Oxidative exoenzymes (OE) of the lignin-degrading enzyme system (LDS) of fungi catalyse the oxidation of a wide range of toxic pollutants. However, very little evidences on fungal degradation or biotransformation of EHCO have been reported, which contain high amounts of asphaltenes and its biodegradation rate is very limited. The aims of this work were to study the ability of Pestalotiopsis palmarum BM-04 to synthesize OE, its potential to biotransform EHCO and to survive in extreme environmental conditions. Enzymatic studies of the LDS showed the ability of this fungus to overproduce high amounts of laccase (LACp) in presence of wheat bran or lignin peroxidase (LIPp) with EHCO as sole carbon and energy source (1300 U mgP−1 in both cases). FT-IR spectroscopy with Attenuated Total Reflectance (ATR) analysis showed the enzymatic oxidation of carbon and sulfur atoms in both maltenes and asphaltenes fractions of biotreated EHCO catalysed by cell-free laccase-enriched OE using wheat bran as inducer. UV-visible spectrophotometry analysis revealed the oxidation of the petroporphyrins in the asphaltenes fraction of biotreated EHCO. Tolerance assays showed the ability of this fungus to grow up to 50 000 p.p.m. of EHCO and 2000 mM of NaCl. These results suggest that P. palmarum BM-04 is a hopeful alternative to be used in remediation processes in extreme environmental conditions of salinity and EHCO contamination, such as the drilling waste from the OOB.

Biocorrosion, as well as the biodeterioration of crude oil and its derivatives, is one of the major environmental, operational and economic problems in the Venezuelan oil industry. Fungal contaminants are able to produce large quantities of biomass and synthesize peroxides and organic acids, causing severe damage on metal surfaces and promoting the contamination and biodeterioration of fuels. No evidences regarding fungal strains have been reported to be associated to petroleum naphtha, widely used as a diluent of extra heavy crude oil (EHCO) in the exploitation processes of the Orinoco Oil Belt, the biggest proven reserve of EHCO worldwide. The aims of this paper were to isolate and identify fungal strains from the naphtha storage tank and the naphtha distribution network from an oil field operator in Venezuela. The results showed the isolation of four different fungal strains. The molecular identification by 28S rRNA sequencing and phylogenetic tree analysis allowed us to identify the presence of: 1) a new uncultured Ascomycota fungus species BM-103, with high identity to novel hyphomycetes Noosia banksiae and Sporidesmium tengii, in the naphtha storage tank; 2) two yeasts, Rhodotorula mucilaginosa BM-104 (Phylum Basidiomycota) and Wickerhamia sp. BM-105 (Phylum Ascomycota), in a highly damaged naphtha pipeline branch and; 3) Cladosporium cladosporioides BM-102 (Phylum Ascomycota) in a cluster oil well. DNA fingerprinting analysis using ERIC-PCR primers pairs also allowed us to detect the presence of R. mucilaginosa BM-104 right in the access of the studied naphtha system. Interestingly, R. mucilaginosa and C. cladosporioides were previously reported as predominant fungal contaminants of diesel and jet fuel and of kerosene and fuel storage systems, respectively. This paper represents the first evidence of fungal strains isolated and identified from the naphtha systems in the Venezuelan oil industry. The results obtained are discussed. L. Naranjo et al.144

Palabras clave: hidrocarburos, índice integral de fitotoxicidad, germinación, plantas RESUMEN La contaminación de ecosistemas por hidrocarburos tóxicos y carcinogénicos derivados del petróleo es un problema ambiental. Por ello, se han desarrollado técnicas como la biorremediación para sanear lugares contaminados utilizando microorganismos con capacidad de degradar hidrocarburos; sin embargo, algunos pueden biotransformar estos compuestos generando metabolitos más tóxicos. Con el objeto de detectar la posible formación de compuestos tóxicos se han diseñado pruebas de toxicidad sobre la germinación y el crecimiento de plántulas de Lactuca sativa, como marco de referencia. La finalidad del presente trabajo fue estudiar el efecto tóxico o estimulante de tres hidrocarburos aromáticos policíclicos: fenantreno, naftaleno y pireno; el efecto del crudo extrapesado Carabobo y sus fracciones de saturados y aromáticos, y finalmente la evaluación del posible efecto tóxico de hidrocarburos biotratatados con Penicillium aculeatum (BM-83). Se demostró que bajas concentraciones de naftaleno, fenantreno, pireno y saturados, generaron un estímulo de crecimiento en las plántulas de L. sativa. Por otro lado, a altas concentraciones de hidrocarburos, el crecimiento del hipocótilo fue el parámetro más afectado, lo que sugiere la importancia de utilizar un índice de toxicidad modificado denominado índice integral de fitotoxicidad (IIF), el cual considera el crecimiento del hipocótilo. El orden de toxicidad de los hidrocarburos según el índice IIF fue el siguiente: naftaleno (100) > fenantreno (65) > pireno (64) > aromáticos (27) > CEP (7) > saturados (1). El IIF permitió evidenciar que el hongo BM-83 tuvo la capacidad de biotransformar los hidrocarburos sin generar compuestos más tóxicos que los originales.Key words: hydrocarbons, integral index of phytotoxicity, germination, plants ABSTRACTThe pollution of ecosystems by toxic and carcinogenic hydrocarbons derived from petroleum is an environmental problem. Therefore, scientists have developed bioremediation Rev. Int. Contam. Ambie. 34 (1) 79-91, 2018

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