Combined action of a bacterial monooxygenase and a fungal laccase for the biodegradation of mono- and poly-aromatic hydrocarbons Antonella Gullotto a , Sergio Branciamore a , Ilaria Duchi a , Maria Francisca Pareja Caño a , Demetrio Randazzo a , Silvia Tilli a , Paola Giardina b , Giovanni Sannia b , Andrea Scozzafava a , Fabrizio Briganti a, * a Laboratorio di Chimica Bioinorganica, Dipartimento di Chimica, Università di Firenze, Via Della Lastruccia 3, 50019 Firenze, Italy b Dipartimento di Chimica Organica e Biochimica, Università di Napoli ‘‘Federico II”, Italy article info Article history: Received 24 October 2007 Received in revised form 22 February 2008 Accepted 28 February 2008 Available online 14 April 2008 Keywords: Toluene Naphthalene Laccase Monooxygenase Biodegradation abstract The combined action of a wide substrate range toluene o-xylene monooxygenase from Pseudomonas sp. OX1, able to convert many aromatic compounds into mono- and di-hydroxylated derivatives, and fungal laccases from Pleurotus ostreatus which oxidize these hydroxylated products yielding polymers with reduced toxicity is described. This strategy permits to overcome many of the substrate specificity prob- lems and dead end toxic products formation generally encountered in complex bacterial biodegradation pathways. Toluene and naphthalene degradations were tested as representative of mono- and poly-aro- matic pollutants. The combined biological action was optimized in micellar and microemulsion systems able to increase the bioavailability of the hydrophobic aromatic pollutants. This approach allows efficient hydroxylations of hydrophobic substrates thus favoring the further action of fungal oxidases. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Nowadays the need of ecosystem conservation and the prob- lems related to the widely diffused environmental pollution evi- denced the necessity to optimize remediation processes for a large variety of toxic xenobiotics continuously dispersed into the environment. Such contaminations can be long-term and have a significant impact on decomposition processes and nutrient cy- cling resulting in environmental problems in groundwater (Musz- kat et al., 1993). Among the most abundant environmental pollutants, the aromatic compounds are of major concern because of their persistence and toxicity (Zedeck, 1980). Soil microorganisms have been extensively characterized in their capabilities to enhance the breakdown of xenobiotic com- pounds (Hollender et al., 2003). The large genetic plasticity and metabolic versatility of microorganisms allow them to acquire the necessary catabolic abilities to use also recalcitrant aromatic compounds as their sole carbon and energy source, but the contin- uing persistence of some xenobiotics in the environment indicates that their biodegradative capacity is not expressed completely or effectively (Sayler, 1991). Developments in molecular biology have the potential to produce organisms with new combinations of traits that can remediate previously un-biodegradable compounds (Brazil et al., 1995), or degrade xenobiotics at a higher rate and/or to a greater extent than naturally occurring organisms (Mason et al., 1997). Unfortunately to obtain the complete detoxification of a broad group of pollutants numerous enzymes belonging to a specific catabolic pathway need to be optimized thus rendering this approach difficult to undertake (Ramos et al., 1994). Furthermore the possibility to devise interesting biodegrada- tion processes through the utilization of white rot fungal lignino- lytic enzymes especially for compounds not readily degraded by bacteria, has recently emerged. This ability arises from the produc- tion of extracellular enzymes like peroxidases and laccases, show- ing low specificity and strong oxidative abilities involved in lignin degradation (Levin and Forchiassin, 2001). They can also act on a broad array of organic compounds containing carbon skeletons similar to those found within the lignin polymer, such as many substituted phenols (Field et al., 1993). Isolated fungal oxidases can be generally utilized in bioconversion processes since they are extracellular glycosilated enzymes, stable to different condi- tions, and particularly laccases do not need any expensive co-sub- strate but only molecular oxygen. Unfortunately laccases are not 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.02.054 * Corresponding author. Tel.: +39 0 554573343; fax: +39 0 554573333. E-mail address: fbriganti@unifi.it (F. Briganti). Bioresource Technology 99 (2008) 8353–8359 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech