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VERTIMAR-2005 Symposium on Marine Accidental Oil Spills Hydrocarbon-degrading marine bacteria relevant in the nitrogen cycle Elena GARCÍA-VALDÉS, Zoyla DAVID, Magdalena MULET, Aina CLADERA, Joan MAYOL and Jorge LALUCAT Departament de Biologia and IMEDEA (CSIC-Universitat de les Illes Balears). Campus UIB, 07122 Palma de Mallorca, Spain ([email protected]; [email protected]; [email protected]) ABSTRACT Hydrocarbon-degrading bacterial strains have been isolated from contaminated sand as the result of the Prestige oil spill. Those microorganisms able to denitrify have been selected. These strains, together with other denitrifying degraders, or strains able to grow diazotrophically selected from our culture collection, have been presumptively identified through the partial 16S rDNA sequence. Strains were physiological characterized as the basis for conducting laboratory microcosms experiments, to follow the crude oil degradation and changes in the bacterial community under different environmental conditions. 1. INTRODUCTION Crude oil spills have a great impact on the bacterial communities in natural ecosystems, modifying their composition due to their toxic effect against many of them, and stimulating the growth of hydrocarbon degraders. Bacterial communities are the first to react in front the presence of contaminants. The growth of degraders is usually limited by the amounts of nutrients needed for the catabolism of the hydrocarbons and for the synthesis of new biomass. Oxygen and nitrogen are limiting factors in the biodegradation rates of the contaminants. Bacteria play key roles not only as degraders, but also in the nitrogen cycling of elements in natural ecosystems, acting as nitrogen fixers and as denitrifiers, affecting in this way the biodegradation rates. To gain inside on the impact of the oil spill on the bacterial communities in the coast, two physiological groups of bacteria able to grow aerobically on components of crude oil have been studied in our culture collection of microorganisms and in natural samples taken in the Galicia coast. In this way, models can be designed in the laboratory to assess the role of each physiological group, and we will be able to compare the communities from contaminated and non-contaminated areas in the Galicia coast. Groups of aerobically hydrocarbon-degraders studied were: 1) Bacteria also able to grow anaerobically as denitrifiers. 2) Bacteria able to grow aerobically and diazotrophically, fixing nitrogen. The isolates were characterised for their ability to grow on hydrocarbons and taxonomically by the analysis of the 16S rRNA sequences. 2. RESULTS AND DISCUSION 1. Samples. Sand samples were taken in March 2004 in Lariño beach (Galicia, Spain) highly polluted with crude oil as a consequence of the Prestige oil spill. This part of the coast was not fertilized previously with nutrients, nor bioaugmentation experiments had been performed. 2. Isolation of denitrifying bacteria. A selective oligotrophic medium with KNO 3 as terminal electron acceptor was used for enrichment or direct isolation under anaerobic conditions. Maltose, starch, glycerol and ethylene-glycol at 0.5% were used as substrate. Incubation temperatures were 20 and 30oC. Isolates were tested for denitrification by culture on liquid medium with the same carbon source and Durham tubes to detect the formation of gas from nitrate. Five pure cultures with different colonial morphologies were obtained when the sample was resuspended in Ringer and directly inoculated on plates, without previous enrichment. Four strains were isolated after enrichment in oligotrophic and copiotrophic selective medium for denitrifiers. Twenty-two strains were isolated after previous aerobic enrichment in mineral medium with naphthalene (9) or hexadecane (13) as the only carbon source in oligotrophic medium. VERTIMAR-2005 Symposium on Marine Accidental Oil Spills 3. Presumptive identification. Partial 16S rRNA sequences (400-1000 nucleotides) were determined by standard procedures previously described (Cladera et al., 2004). A BLAST analysis on nucleotide sequences databases, together with the construction of phyogenetic trees, affiliated most of the isolates to described species or genera of marine bacteria. Microbacterium: M oxydans, M. maritypicum Marinobacter Nocardioides simplex Ochrobactrum Oerskovia turbata Rhodococcus Pseudomonas: P. fulva, P. putida, P. pachastrellae The isolates were classified taxonomically and their capacity of growth on hydrocarbons (hexadecane and naphthalene) was also tested for each isolate. 4. Selection of denitrifiers and nitrogen-fixers from culture collection. Previously isolated marine hydrocarbon-degraders of the species Pseudomonas stutzeri able to grow anaerobically with nitrate were selected for further studies. The collection included naphthalene, phenantrene, hexadecane and monoaromatic-degraders. Some of them could grow anaerobically with nitrate on monoaromatics, and others utilized both, aromatics and alcanes aerobically (Song et al., 2000). Strains B1SMN1 and S1MN1 were isolated from a waste water lagoon, and are able to grow diazotrophically on naphthalene, and are independent of combined nitrogen sources (Rosselló et al., 1991). This collection of strains was characterized taxonomically and with respect their denitrification and nitrogen fixation ability. Studies on the fate in microcosms experiments of these autochthonous marine bacteria isolated from the Galicia coast, together with selected culture collection strains, are under way. REFERENCES Cladera AM, A Bennasar, M Barceló, J Lalucat, E García-Valdés (2004) Comparative genetic diversity of Pseudomonas stutzeri genomovars, clonal structure and phylogeny of the species: J.Bacteriol. Vol. 186 nº16 p. 5239-5248. B, Norberto J. Palleroni, Max M. Häggblom (2000) Isolation and characterization of diverse halobenzoate degrading denitrifying bacteria from soils and sediments. Appl. Environ. Microbiol Vol: 66 nº8 p. 3446-3453. Rosselló R, E García-Valdés, J Lalucat, J Ursing (1991) Genotypic and phenotypic diversity of Pseudomonas stutzeri. System. Appl. Microbiol. 14, p.150-157. Acknowledgements This work was supported by grant VEM2003-20565/INTER from the CICYT, Spain. We thank NJ Palleroni for supplying bacterial strains. Researchers of the Instituto de Investigaciones Marinas (CSIC-Vigo) are acknowledged for helping in deciding the sampling area.