ORIGINAL ARTICLE Isolation of Gram-positive n-alkane degraders from a hydrocarbon-contaminated Mediterranean shoreline P. Quatrini 1 , G. Scaglione 1 , C. De Pasquale 2 , S. Riela 3 and A.M. Puglia 1 1 Dipartimento di Biologia Cellulare e dello Sviluppo ‘‘A. Monroy’’, University of Palermo, Palermo, Italy 2 Centro Interdipartimentale per lo Studio dell’Ecologia degli Ambienti Costieri, University of Palermo, Palermo, Italy 3 Dipartimento di Chimica Organica, ‘‘E. Paterno ` ’’, University of Palermo, Palermo, Italy Introduction Petroleum hydrocarbon (HC)-degrading micro-organisms are widespread and most HC catabolic pathways have been elucidated (van Hamme et al. 2003). Nevertheless oil and oil-derived compounds are still the most threaten- ing contaminants in the environment. Understanding the microbial ecology and the constraints on biodegradation of oil-contaminated sites in different environments would help in finding the best degrading conditions to favour complete environmental clean-up. Bioremediation has often been proposed as a cost- effective, efficient alternative to physicochemical treat- ments (Atlas 1991). Bioaugmentation (introduction of selected degraders in the contaminated site) and biosti- mulation (based on catabolic ability of the autochthonous microflora, stimulated by fertilization and ⁄ or by aeration) have both been used in field and microcosm trials with success (Kaplan and Kitts 2004; Heinaru et al. 2005; Stall- wood et al. 2005). Using culture-independent and isolation techniques, different microbial communities and bacterial groups have been characterized from HC-contaminated sites, in different geographical and ecological contexts (Cho et al. 1997; Margesin et al. 2003; Kasai et al. 2005; Yakimov et al. 2005; Brakstad and Bonaunet 2006). Although dif- ferent conditions and different periods are considered in each work, some general trends indicate that Gram-nega- tive Proteobacteria (Pseudomonas, Alkanivorax and related genera) and CFB group (Cytophaga, Flavobacterium, Bac- teroides) are often dominant in microcosms after oil spill simulations (MacNaughton et al. 1999) and in HC-con- taminated environments after biostimulation (Kaplan and Kitts 2004; Ro ¨ling et al. 2004). These groups of bacteria are associated with a first fast petroleum-degradation phase, and their abundance was positively correlated to Keywords actinomycetes, alkB, GC-MS analysis, Gordonia, n-alkane degradation, Nocardia, Rhodococcus. Correspondence Paola Quatrini, Dipartimento di Biologia Cellulare e dello Sviluppo, University of Palermo, Edificio 16, Viale delle Scienze, 90128 Palermo, Italy. E-mail: quatrini@unipa.it 2007 ⁄ 0458: received 24 March 2007, revised 22 June 2007 and accepted 19 July 2007 doi:10.1111/j.1365-2672.2007.03544.x Abstract Aims: To investigate the petroleum hydrocarbon (HC)-degrading potential of indigenous micro-organisms in a sandy Mediterranean coast, accidentally con- taminated with petroleum-derived HCs. Methods and Results: Using culturable methods, a population of Gram-posi- tive n-alkane degraders was detected in the contaminated soil. Five isolates, identified as one Nocardia, two Rhodococcus and two Gordonia strains, were able to degrade medium- and long-chain n-alkanes up to C 36 as assessed by growth assays and gas chromatography-mass spectrometry analysis. Diverging alkane hydroxylase-encoding genes (alkB) were detected by PCR, using degene- rated primers, in all the strains; multiple sequences were obtained from the Nocardia strain, while only one alkB gene was detected in the Rhodococcus and Gordonia strains. The majority of the alkB sequences were related to Rhodococ- cus alkB2, but none was identical to it. Conclusions: Actinomycetes might have a key role in bioremediation of n-alkane-contaminated sites under dry, resource-limited conditions, such as those found in the Mediterranean shorelines. Significance and Impact of the Study: To our knowledge, this is the first study on the bioremediation potential in Mediterranean contaminated beaches. Journal of Applied Microbiology ISSN 1364-5072 ª 2007 The Authors Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 251–259 251