.. ' Biodegradation (2008) 19:425-434 DOI 10.1007/510532-007-9148-0 A microcosm system and an analytical protocol to assess PAH degradation and metabolite formation in soils :r¿daArias ~ Jorge Bauzá . Joana TobelIa . Joaquim Vila . Magdalena GrifolI Received: 18 April 20071 Accepted: 10 August 20071 PubJished online: J2 September 2007 @ Springer Science+Business ~1edia B.V. 2007 Abstract During bioremediation of polycyclic aromatic hydrocarbon (PAH)-polluted soils accumu- lation of polar metabolites resulting from the biological activity may occur. Since these polar metabolites are potentially more toxic than the parental products, a better understanding of the processes in volved in the production and fate of these oxidation products in soil is needed. In the present work we describe the design and set-up of a static soil microcosm system and an analytical methodology for detection of PAHs and their oxidation products in soils. When applied to a soil contaminated with phenanthrene, as a model PAH, and l-hydroxy- 2-naphthoic acid, diphenic acid, and phthalic ~cid as putative metabolites, the extraction and fractionation procedures resulted in recoveries of 93%, 89%, 100%, and 89%, respectively. The application of the standardized system to study the biodegradation of phenanthrene in an agricultural soil with and ~ithout inoculation of the high molecular weight PAH-degrading strain Mycobacterium sp. API, demonstrates its suitability for determining the envi- ron mental fate of PAHs in polluted soils and for L. Arias. J. Bauzá . J. Tobella .J. Vila . M. Grifoll (121) Department of Microbiology, University of Barcelona, Diagonal 645, Barcelona 08028, Spain e-mail: mgrifoll@ub.edu (L. AriaS' 'o Mic~obiology ~chool, University of ~ntioquia,27tl1 st(Fet ~ 53-J08,.Medellin, CA 1226, USA ~j -. evaluating the effect of bioremediative treatments. In inoculated microcosms 35% of the added phenan- threne was depleted, 19% being recovered as CO 2 and 3% as diphenic acid. The latter, together with other two unidentified metabolites, accumulated in soil. Keywords Biodegradation. Polycyclic aromatic hydrocarbons . Soil microcosms . Phenanthrene . Bacterial PAH metabolites Introduction Characterization ofbiodegradation processes in soils is a key issue in determining the fate of contaminants in polluted soils and should provide valuable information for the design, optimization and risk assessment of decontamination treatments. PAHs are pollutants of special concem due to thejr ubiquitous presence in soils contaminated with oil and coal derivatives, their persistence, and potential toxicity/carcinogenicity (Kanaly and Harayama 2000). Biodegradation routes for 2- and 3-ring PAHs by soil bacteria that use them as asole source of carbon and energy are well characterized. In general, the studied Gram-negative strains possess a complete set of enzymes for the total assimilation/mineralization of those compounds (Cemiglia 1992). Soil enrich- ment cultures with four-ring PAHs as asole carbon source usually leads to the isolation of actinobacteria, ~ Springer