Short communication Shewanella putrefaciens in a fuel-in-water emulsion from the Prestige oil spill

Microorganisms that colonize the fuel-in-water emulsion from the Prestige spill have been compared with those from Exxon-Valdez. Both emulsions contained non-fermentative gram-negative rods but unlike Exxon-Valdez’s, the Prestige’s spill contained anaerobic bacteria and no fungi. Our main finding has been the identification of Shewanella putrefaciens, a bacterium promising for bioremediation. The identification of the key micro-organisms which play a role in pollution processes from petroleum tanker spills is relevant both for the prevention and control of infectious diseases in aquatic organisms and the development of optimal in situ bioremediation strategies. In this paper we report our efforts to characterize bacterial communities in a fuel-water emulsion from Prestige’s spill. Samples were collected from some beaches in San Vicente de la Barquera Cantabria, Spain following the arrival of great spots of emulsion that came in from the 7 to 10 December 2002 to the North coast of Spain. Chemical studies showed that the samples, with the shape of black mousse chapapote , were aged petroleum in the form of fuel-in-water emulsion and with the composition previously reported for the Prestige tanker’s spill Table 1 . For microbiological study, the samples were collected in sterilized glass bottles which were filled up to overflow with the emulsion, hermetically closed and stored at room temperature. The emulsion was sown on agar-blood plates and other media for bacterial and fungal culture. At 24 hours of culture, growth of bacterial colonies was observed but not fungi. We used the Vitek-2 system BioMérieuxVitek to identify and analyze bacteria. This system contains microformatted reagent cards for biochemical tests and selective growth media. Also, the Vitek-2 system automates the front-end sample processing including initial inoculum dilution, density verification, card-filling and carding-sealing steps. The instrument transfers cards to the reader-incubator and ejects them into a disposal bin at the completion of testing. The results provided by this identification system were correlated by us with basic characteristics such as macroscopic morphology and Gram staining results. The bacterial flora which was identified in the chapapote is given in Table 2. As a reference, we report the microbial flora characterized in a similar polluted environment a beach in Alaska as a result of Exxon-Valdez’s spill. Both emulsions, from Prestige and Exxon-Valdez spills contained non-fermentative gram-negative rods but only the Prestige’s spill contained anaerobic bacteria and no fungi. Among the species that we have characterised we would like to emphasize the presence of the gammaAntonie van Leeuwenhoek 86: 283–285, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 283 XML — ms-code anto43r2 — PIPS 5274285 — 11 Oct 2004 — Grafikon 203006016 proteobacteria Shewanella and, in particular, Shewanella putrefaciens. This bacterium is implicated in oil pipeline and tank corrosion, formation of biofilms Bagge et al. 2001 , fish spoilage, bioremediation of halogenated hydrocarbons and oil-contaminated environments Petrovskis et al. 1994; Kim et al. 1999, 2000 and destructive souring of crude petroleum Semple et al. 1987 . Their main interest arises from their ability to couple the reduction of Fe III with the complete oxidation to CO2 of petroleum components under the anoxic conditions prevalent in polluted sites. After oil spills, the structure and diversity of the dominant community changes substantially with time: from primarily eukaryotic biomass to gramnegative bacterial biomass. Oil treatment with fertilizers encourages the growth of gram-negative microorganisms within the alpha-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum MacNaughton et al. 1999 . A difference between the microbial flora from the Prestige and Exxon-Valdez spills is the presence of Bacteroides in the first and Cytophaga in the later. We consider that this feature is due to ecological differences and mainly to changes in nutrients and temperature from marine environments as different as Finisterre continental and Alaska sub-arctic . With regard to the characterisation of Shewanella, it has been reported that Shewanella alga is the predominant human clinical isolate 77% , while S. putrefaciens represents the majority of nonhuman isolates 89%, Khashe et al. 1998 . The finding of the reported Shewanella isolates in a non clinical environment is consistent with its identification as S. putrefaciens. Our isolates grew at 37oC which allowed us to distinguish S. putrefaciens from the new species Shewanella baltica, proposed by Höfle et al. 2000 for strains which have been isolated mostly from the Baltic Sea and which grow at 4 oC. Table 1. Chemical composition of the petroleum from Prestige, Erika, Baltic Carrier and Exxon-Valdez spills Bayona 2002; CEDRE 2002; Oudot 2002 Saturates hydrocarbons % Aromatics hydrocarbons % Resins % Asphaltenes % Prestige tanks 26.6 52.8 8.4 12.2 Prestige emulsion 24.6 50.2 9.9 14.2 Erika 22.2 55.6 15.6 6.6 Baltic Carrier 40.9 37.9 11.5 9.7 Exxon-Valdez* 75.0 15.0 6.1 4.0 * Unknown. Could not be found after an intense bibliographic research. Instead, we report the results of an analysis from the Alaska North Slope crude oil made recently by Environment Canada courtesy of J. W. Short, Auke Bay Laboratory, Alaska Fisheries Science Center, National Marine Fisheries Service, NOAA, 11305 Glacier Highway . Table 2. Bacterial and fungi communities found in fuel-in-oil emulsions from the Prestige’s spill and those that showed efficient degradation of crude oil in the coast of Alaska, after Exxon-Valdez’s spill whose presence was enhanced more than 10.000 times than usual Prestige ́s spill Exxon-Valdez ́s spill Chanelli, 1991 Aerobic gram-negative rods Aeromonas caviae Enterobacter amnigenus Klebsiella pneumoniae Proteus mirabilis Vibrio sp Non-fermentative gram-negative rods Shewanella putrefaciens Pseudomonas fluorescens Pseudomonas stutzeri Pseudomonas vesicularis