Microbial diversity in coastal subsurface sediments: a cultivation approach using various electron acceptors and substrate gradients

Abstract

Microbial communities in coastal subsurface sediments are scarcely investigated and have escaped attention so far. But since they are likely to play an important role in biogeochemical cycles, knowledge of their composition and ecological adaptations is important. Microbial communities in tidal sediments were investigated along the geochemical gradients from the surface down to a depth of 5.5 m. Most-probable-number (MPN) series were prepared with a variety of different carbon substrates, each at a low concentration, in combination with different electron acceptors such as iron and manganese oxides. These achieved remarkably high cultivation efficiencies (up to 23% of the total cell counts) along the upper 200 cm. In the deeper sediment layers, MPN counts dropped significantly. Parallel to the liquid enrichment cultures in the MPN series, gradient cultures with embedded sediment subcores were prepared as an additional enrichment approach. In total, 112 pure cultures were isolated; they could be grouped into 53 different operational taxonomic units (OTU). The isolates belonged to the Proteobacteria, "Bacteroidetes," "Fusobacteria," Actinobacteria, and "Firmicutes." Each cultivation approach yielded a specific set of isolates that in general were restricted to this single isolation procedure. Analysis of the enrichment cultures by PCR and denaturing gradient gel electrophoresis revealed an even higher diversity in the primary enrichments that was only partially reflected by the culture collection. The majority of the isolates grew well under anoxic conditions, by fermentation, or by anaerobic respiration with nitrate, sulfate, ferrihydrite, or manganese oxides as electron acceptors.

FIG. 1.

Depth profiles of sulfate (•) and chloride (□) and major lithological facies in sediments from the Gröninger Plate (left) and Neuharlingersieler Nacken (right) sites.

FIG. 2.

Relative contributions of the different phylogenetic groups to the numbers of isolates present in the culture collection obtained from different depths at the Neuharlingersieler Nacken and Gröninger Plate sites. The numbers above the columns indicate depth in centimeters, and the numbers on the left indicate the relative contributions to the culture collection from the respective depths.

FIG. 3.

Comparison of the different enrichment cultures and the microbial community in situ (original sediments [sed.]) by PCR and DGGE using primers specific to Eubacteria. Enrichment cultures in gradient tubes with carboxylic and amino acids are labeled “Grad. TCA”; those with alcohols and fatty acids are labeled “Grad. ALC.” Bands that were excised for sequence analysis are numbered consecutively, beginning at the upper edge of the gel. Arrowheads indicate only those DGGE bands that gave reliable results after excision and sequence analysis.