In November 2002, the oil tanker Prestige sank off the Spanish coast after releasing approximately 17,000 tones of heavy fuel, coating several hundred kilometers of coastline in oil sludge. In December 2002 and February 2003, samples were collected from the shore of the Galician coast to analyse the indigenous population ability to carry out crude oil degradation in situ. Carbon isotopic ratio of the dissolved inorganic carbon (DIC) in seawater samples was used as a rapid method to directly assess activity of microbes on the oil components. 12CO2/13CO2 ratio in samples from certain locations along the coast revealed degradation of a very delta13C-negative source such as the Prestige crude oil (-30.6 per thousand). Putative biodegradation processes taking place at areas with high income of fresh seawater could not be detected with this technique. Laboratory-scale biostimulation processes carried out in samples with the highest oil biodegradation activity showed that N/P deficiency in seawater is a limiting factor for crude oil degradation. The most probable number (MPN) of crude oil component degraders was estimated for several aromatic compounds (naphthalene, anthracene, phenanthrene, pyrene) and for undecane. Our results clearly show that bacteria present in the contaminated water are readily able to transform components of the crude oil into inorganic carbon.

Azoarcus anaerobius, a strictly anaerobic, gram-negative bacterium, utilizes resorcinol as a sole carbon and energy source with nitrate as an electron acceptor. Previously, we showed that resorcinol degradation by this bacterium is initiated by two oxidative steps, both catalyzed by membrane-associated enzymes that lead to the formation of hydroxyhydroquinone (HHQ; 1,2,4-benzenetriol) and 2-hydroxy-1,4-benzoquinone (HBQ). This study presents evidence for the further degradation of HBQ in cell extracts to form acetic and malic acids. To identify the A. anaerobius genes required for anaerobic resorcinol catabolism, a cosmid library with genomic DNA was constructed and transformed into the phylogenetically related species Thauera aromatica, which cannot grow with resorcinol. By heterologous complementation, a transconjugant was identified that gained the ability to metabolize resorcinol. Its cosmid, designated R ؉ , carries a 29.88-kb chromosomal DNA fragment containing 22 putative genes. In cell extracts of T. aromatica transconjugants, resorcinol was degraded to HHQ, HBQ, and acetate, suggesting that cosmid R ؉ carried all of the genes necessary for resorcinol degradation. On the basis of the physiological characterization of T. aromatica transconjugants carrying transposon insertions in different genes of cosmid R؉ , eight open reading frames were found to be essential for resorcinol mineralization. Resorcinol hydroxylase-encoding genes were assigned on the basis of sequence analysis and enzyme assays with two mutants. Putative genes for hydroxyhydroquinone dehydrogenase and enzymes involved in ring fission have also been proposed. This work provides the first example of the identification of genes involved in the anaerobic degradation of aromatic compounds by heterologous expression of a cosmid library in a phylogenetically related organism.