Microbial communities in the chemocline of a hypersaline deep-sea basin (Urania basin, Mediterranean Sea)

Abstract

The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea. Since this basin is located at a depth of approximately 3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon. In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated. The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface. Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated. Employing 11 cultivation methods, we isolated a total of 70 bacterial strains. The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods. These strains were identified as flavobacteria, Alteromonas macleodii, and Halomonas aquamarina. All 70 strains could grow chemoorganoheterotrophically under oxic conditions. Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically. While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unusually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source. Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.

FIG. 1

Locations of the Urania basin and other hypersaline brine lakes in the eastern Mediterranean Sea.

FIG. 2

Physicochemical parameters, total cell counts, and MPN across the oxic-anoxic interface of the Urania basin. (A) Oxygen and sulfide concentrations; (B) sulfate, ortho-phosphate, and chloride concentrations; (C) total cell counts; (D) viable cell counts in anoxic media containing the amino acid mixture (solid bars) and in oxic media containing thiosulfate (cross-hatched bars). Only values for the media which yielded maximum MPN are shown. The percentages are percentages relative to the total cell count. The error bars indicate standard deviations.

FIG. 3

Extracellular β-glucosidase, leucine aminopeptidase, and alkaline phosphatase activities in the Urania basin. The error bars indicate standard deviations.

FIG. 4

Comparison of 16S rDNA fragments of the strains isolated with fingerprints of the natural bacterial community in the chemocline by DGGE fingerprinting. Negative images of ethidium bromide-stained gels are shown. (A) Anaerobically isolated strains; (B) aerobically isolated strains. Strains were assigned to species and to genera if sequence identities were >97 and >93%, respectively. Strains marked by shading had 16S rDNA sequences identical to those of band A1, A2, or A7. CFB-group, Cytophaga-Flexibacter-Bacteroides group.

FIG. 5

Phylogenetic affiliations of the strains isolated and the molecular isolates from the chemocline bacterial community. The tree is based on 452 informative nucleotide positions of the 16S rRNA gene. Chlorobium limicola was used as the outgroup.

FIG. 6

Genetic diversity of the three groups of bacterial isolates which represent numerically significant phylotypes. The dendrograms are based on ERIC PCR band patterns. CF-group, Cytophaga-Flavobacterium group.

FIG. 7

Diversity of phylotypes in the collection of 70 bacterial isolates. A total of 13 phylotypes were detected. After the order of strains was randomized, sequential detection of phylotypes was plotted in a cumulative manner (●). For comparison, a rarefaction curve (solid line) was produced from the data by employing the freeware program Analytical Rarefaction 1.2 (compiled by Steven M. Holland; available at http://www.uga.edu/∼strata/Software.html). The dotted lines indicate 95% confidence intervals.