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The diversity and dynamics of a bacterial community extracted from an exploited oil field with high natural soil salinity near Comodoro Rivadavia in Patagonia (Argentina) were investigated. Community shifts during long-term incubation with diesel fuel at four salinities between 0 and 20% NaCl were monitored by singlestrand conformation polymorphism community fingerprinting of the PCR-amplified V4-V5 region of the 16S rRNA genes. Information obtained by this qualitative approach was extended by flow cytometric analysis to follow quantitatively the dynamics of community structures at different salinities. Dominant and newly developing clusters of individuals visualized via their DNA patterns versus cell sizes were used to identify the subcommunities primarily involved in the degradation process. To determine the most active species, subcommunities were separated physically by high-resolution cell sorting and subsequent phylogenetic identification by 16S rRNA gene sequencing. Reduced salinity favored the dominance of Sphingomonas spp., whereas at elevated salinities, Ralstonia spp. and a number of halophilic genera, including Halomonas, Dietzia, and Alcanivorax, were identified. The combination of cytometric sorting with molecular characterization allowed us to monitor community adaptation and to identify active and proliferating subcommunities.Many oil fields are located in semiarid regions characterized by soils of high natural salinity. Careless operation during oil extraction and processing contaminates the soil, for instance, via the generation of large volumes of oily and saline wastewater. Strategies are thus needed to effectively treat oil pollution of highly saline waters and soils.The degradation of hydrocarbons as major components of mineral oil is generally brought about by microorganisms. At the salinity of seawater (ϳ3% total salts, mainly NaCl) or below, most low-molecular-weight components of mineral oil are easily degraded by a variety of microorganisms. However, the range of degrading organisms decreases with increasing salinity. There are only a few reports on the degradation of hydrocarbons in hypersaline environments. Enrichment cultures from the Great Solar Lake grew on mineral oil at salinities up to 17.2% (70), and a bacterial consortium and an extremely halophilic archaeon grew on hexadecane and crude oil at salinities of 15% and 32%, respectively (5). Degradation of hydrocarbons (mainly n-alkanes) by archaea (Halobacteria) at 15 to 32% NaCl was also reported (40). An extremely halophilic archaeon from a salt marsh degraded various saturated and aromatic hydrocarbons at salinities of up to 31% and grew optimally at 22% NaCl (10). In our own laboratory, microbial communities from saline soils in Patagonia degraded diesel fuel up to a salinity of 17.5% (59). Although some members of the communities were identified, their contributions to the diesel fuel biodegradation remained obscure.Information on the phylogenetic diversity of microbial communities can be obtained by molecular methods, like...

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Microbial communities from three Argentinean saline soils were extracted and tested for their ability to degrade diesel fuel in liquid culture at salinities between 0% and 25%. In each case, the degradation process was continuously monitored by measuring oxygen consumption. Two communities (CR1 and CR2) showed nearly equal degrees of degradation across a salinity range of 0%-10% (the former degrading about 63% of the diesel fuel and the latter about 70% after 53 and 80 d, respectively). Furthermore, the degree of degradation was not significantly lower in the presence of 17.5% salt (58% and 65% degraded, respectively). A third community (El Zorro) showed a maximum turnover at 5% salt (79% diesel fuel degraded) and significant degradation (66%) at a salinity of 10%. However, the degree of degradation by this community clearly dropped at 0% and 15% salt. None of the communities were able to degrade diesel fuel in the presence of 25% salt, but the living cell counts showed that components of the microbial population survived the long-term exposure. The surviving portion is obviously sufficient to allow substantial restoration of the original community, as verified by the BIOLOG method. Isolates of the CR1 community were identified as members of the genera Cellulomonas, Bacillus, Dietzia, and Halomonas. In light of our investigations, the bioremediation of contaminated saline soils should be quite possible if the salinity of the soil water is lower than 15% or if it is reduced below this limit by the addition of water.

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The residual fractions remaining after microbial degradation of diesel fuel, different deparaffinized raffinates and extracts from long-term contaminated soils were analyzed by liquid chromatography, gas chromatography, infrared spectrometry and mass spectrometry. The quantity of saturated hydrocarbons decreased after the microbial treatment, whereas the portion of polar compounds increased. The total content of aromatics changed only insignificantly. n-Paraffins < C26 were found to be no longer present in mineral oils degraded to exhaustion. Infrared spectrometry revealed oxygen compounds in the residues, mainly ketones, fatty acids and esters. Elementary analysis confirms the presence of nitrogen, oxygen and sulphur compounds in the degraded products. The gas chromatograms of high boiling oils, as well as of residues and extracts, consist mainly of a large base "envelope" (about 95% of the total area); thus gc/ms coupling reaches the limits of its applicability. However, mass spectrometry with direct inlet gives valuable information regarding hydrocarbon type analysis. The results revealed the preferable degradation of alkanes, 1-ring aliphatics and benzenes and an enrichment of condensed cycloaliphatics and aromatics. The latter compounds are known to be resistant to microbial attack.

The microbial activity of pristine and contaminated soils was investigated by measuring the following parameters: glucose induced respiration, dimethylsulphoxide reduction and the hydrolysis of fluorescein diacetate. The viable counts were determined by the plate count method. The ability of the autochthonous microorganisms of the investigated soils to degrade diesel fuel was determined in a closed system on the basis of the oxygen consumption and by direct measurements of the hydrocarbon concentrations. As expected, compost showed the highest microbial activity with regard to all three parameters, followed by the grassland and the arable soil samples which were also found to have high activity. However, soils that had been exposed to mineral oil for a long period of time showed significantly lower values. Microorganisms from contaminated sites had a high degradation potential; few pristine soils reached similar turnover rates. The investigations showed that the level of the degradation of diesel fuel in pristine soils correlated with their microbial activity, but this correlation was not found in the investigated contaminated soils.