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Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill.

ABSTRACT: The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.

SUBMITTER: Mulet M

PROVIDER: S-EPMC3028729 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill.

Mulet Magdalena M David Zoyla Z Nogales Balbina B Bosch Rafael R Lalucat Jorge J García-Valdés Elena E

Applied and environmental microbiology 20101203 3

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial en ...[more]

PMID: 21131512

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Project description:The bacterial communities in two different shoreline matrices, rocks and sand, from the Costa da Morte, northwestern Spain, were investigated 12 months after being affected by the Prestige oil spill. Culture-based and culture-independent approaches were used to compare the bacterial diversity present in these environments with that at a nonoiled site. A long-term effect of fuel on the microbial communities in the oiled sand and rock was suggested by the higher proportion of alkane and polyaromatic hydrocarbon (PAH) degraders and the differences in denaturing gradient gel electrophoresis patterns compared with those of the reference site. Members of the classes Alphaproteobacteria and Actinobacteria were the prevailing groups of bacteria detected in both matrices, although the sand bacterial community exhibited higher species richness than the rock bacterial community did. Culture-dependent and -independent approaches suggested that the genus Rhodococcus could play a key role in the in situ degradation of the alkane fraction of the Prestige fuel together with other members of the suborder Corynebacterineae. Moreover, other members of this suborder, such as Mycobacterium spp., together with Sphingomonadaceae bacteria (mainly Lutibacterium anuloederans), were related as well to the degradation of the aromatic fraction of the Prestige fuel. The multiapproach methodology applied in the present study allowed us to assess the complexity of autochthonous microbial communities related to the degradation of heavy fuel from the Prestige and to isolate some of their components for a further physiological study. Since several Corynebacterineae members related to the degradation of alkanes and PAHs were frequently detected in this and other supralittoral environments affected by the Prestige oil spill along the northwestern Spanish coast, the addition of mycolic acids to bioremediation amendments is proposed to favor the presence of these degraders in long-term fuel pollution-affected areas with similar characteristics.

Project description:Availability of inorganic nutrients, particularly nitrogen and phosphorous, is often a primary control on crude oil hydrocarbon degradation in marine systems. Many studies have empirically determined optimum levels of inorganic N and P for stimulation of hydrocarbon degradation. Nevertheless, there is a paucity of information on fundamental kinetic parameters for nutrient enhanced crude oil biodegradation that can be used to model the fate of crude oil in bioremediation programmes that use inorganic nutrient addition to stimulate oil biodegradation. Here we report fundamental kinetic parameters (Ks and qmax) for nitrate- and phosphate-stimulated crude oil biodegradation under nutrient limited conditions and with respect to crude oil, under conditions where N and P are not limiting. In the marine sediments studied, crude oil degradation was limited by both N and P availability. In sediments treated with 12.5 mg/g of oil but with no addition of N and P, hydrocarbon degradation rates, assessed on the basis of CO2 production, were 1.10 ± 0.03 μmol CO2/g wet sediment/day which were comparable to rates of CO2 production in sediments to which no oil was added (1.05 ± 0.27 μmol CO2/g wet sediment/day). When inorganic nitrogen was added alone maximum rates of CO2 production measured were 4.25 ± 0.91 μmol CO2/g wet sediment/day. However, when the same levels of inorganic nitrogen were added in the presence of 0.5% P w/w of oil (1.6 μmol P/g wet sediment) maximum rates of measured CO2 production increased more than four-fold to 18.40 ± 1.04 μmol CO2/g wet sediment/day. Ks and qmax estimates for inorganic N (in the form of sodium nitrate) when P was not limiting were 1.99 ± 0.86 μmol/g wet sediment and 16.16 ± 1.28 μmol CO2/g wet sediment/day respectively. The corresponding values for P were 63 ± 95 nmol/g wet sediment and 12.05 ± 1.31 μmol CO2/g wet sediment/day. The qmax values with respect to N and P were not significantly different (P < 0.05). When N and P were not limiting Ks and qmax for crude oil were 4.52 ± 1.51 mg oil/g wet sediment and 16.89 ± 1.25 μmol CO2/g wet sediment/day. At concentrations of inorganic N above 45 μmol/g wet sediment inhibition of CO2 production from hydrocarbon degradation was evident. Analysis of bacterial 16S rRNA genes indicated that Alcanivorax spp. were selected in these marine sediments with increasing inorganic nutrient concentration, whereas Cycloclasticus spp. were more prevalent at lower inorganic nutrient concentrations. These data suggest that simple empirical estimates of the proportion of nutrients added relative to crude oil concentrations may not be sufficient to guarantee successful crude oil bioremediation in oxic beach sediments. The data we present also help define the maximum rates and hence timescales required for bioremediation of beach sediments.

Dataset Information

Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill.

Publications

Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill.

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