Population dynamics within a microbial consortium during growth on diesel fuel in saline environments

Abstract

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 single-strand 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.

FIG. 1.

Oxygen consumption in two or three replicate samples during the degradation of diesel fuel by a bacterial consortium at different NaCl concentrations. Measurement points (by CFU, total cell counts, BIOLOG, and protein content) after 21, 42, and 84 days are indicated.

FIG. 2.

SSCP fingerprint of the V4-V5 regions of bacterial 16S rRNA genes. Lanes: 1, inoculum (cultivated at 7.5% NaCl); 2, 0% NaCl, 42 days; 3, 0% NaCl, 84 days; 4, 7.5% NaCl, 42 days; 5, 7.5% NaCl, 84 days; 6, 15% NaCl, 42 days; 7, 15% NaCl, 84 days; 8, 20% NaCl, 42 days; 9, 20% NaCl, 84 days. The bands marked by arrows were cut from the gel, and the DNA was extracted, reamplified, and sequenced. When direct sequencing failed, the reamplified DNA was cloned, and a representative of each ARDRA pattern was sequenced. The results are listed in Table 3.

FIG. 3.

Community dynamics during growth at different NaCl concentrations over a time range of 84 days. The population patterns were obtained by determination of bacterial DNA content and cell size. All incubations were started with the same inoculum. Cell cycle-related (circles) and salinity-related (arrows) subpopulations are highlighted; see Results for details.

FIG.4.

Flow cytometric analysis of communities incubated for (A) 42 and (B) 84 days at different NaCl concentrations. DNA-content-versus-forward-scatter behavior was determined, and subcommunities were sorted according to sort gates 1 to 30, as shown. The taxonomic classification of the sequences is based on BLAST and Ribosomal Database Project database retrievals and, in some cases, also on phylogenetic analysis with ARB (data not shown). The bar diagrams show the taxonomic compositions of the subcommunities obtained by sorting and 16S rRNA gene sequencing (for sequencing results, see the supplemental material). The thick arrow in panel B indicates a unique subcommunity that appeared at a proportion of 1%, and the thin arrow indicates another ungated subcommunity after 84 days of incubation.

FIG.4.

Flow cytometric analysis of communities incubated for (A) 42 and (B) 84 days at different NaCl concentrations. DNA-content-versus-forward-scatter behavior was determined, and subcommunities were sorted according to sort gates 1 to 30, as shown. The taxonomic classification of the sequences is based on BLAST and Ribosomal Database Project database retrievals and, in some cases, also on phylogenetic analysis with ARB (data not shown). The bar diagrams show the taxonomic compositions of the subcommunities obtained by sorting and 16S rRNA gene sequencing (for sequencing results, see the supplemental material). The thick arrow in panel B indicates a unique subcommunity that appeared at a proportion of 1%, and the thin arrow indicates another ungated subcommunity after 84 days of incubation.

FIG. 5.

MDS based on taxon abundance data of the subcommunities as derived by flow cytometry and PCR analyses. The symbol sizes indicate the relative abundances (cell counts) of the subcommunities. The numbers beside the symbols are the numbers of the subcommunities as given in Fig. 4.