Detection of abundant sulphate-reducing bacteria in marine oxic sediment layers by a combined cultivation and molecular approach

The depth distribution and diversity of sulphate-reducing bacteria (SRB) was analysed in the upper intertidal zone of a sandy marine sediment of the Dutch island Schiermonnikoog. The upper centimetre of the sediment included the oxic-anoxic interface and was cut into five slices. With each slice, most probable number (MPN) dilution series were set up in microtitre plates using five different substrates. In the deeper sediment layers, up to 1 x 10(8) cm(-3) lactate-utilizing SRB were counted, corresponding to 23% of the total bacterial count. From the highest positive dilutions of the MPN series, 27 strains of SRB were isolated in pure culture. Sequencing of a 580 bp fragment of the 16S rDNA revealed that 21 isolates had identical sequences, also identical with that of the previously described species Desulfomicrobium apsheronum. However, the diversity of the isolates was higher with respect to their physiological properties: a total of 11 different phenotypes could be distinguished. Genomic fingerprinting by enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR) revealed an even higher diversity of 22 different genotypes. A culture-independent analysis by PCR and denaturing-gradient gel electrophoresis (DGGE) revealed that the partial 16S rDNA sequence of the isolated D. apsheronum strains constituted a significant fraction of the Desulfovibrionaceae. The high subspecies diversity suggests that this abundant aggregate-forming species may have evolved adaptations to different ecological niches in the oxic sediment layers.