ABSTRACT
Recently, four Thiomicrospira strains were isolated from a coastal mud flat of the German Wadden Sea (T. Brinkhoff and G. Muyzer, Appl. Environ. Microbiol. 63:3789-3796, 1997). Here we describe the use of a polyphasic approach to investigate the functional role of these closely related bacteria. Microsensor measurements showed that there was oxygen penetration into the sediment to a depth of about 2.0 mm. The pH decreased from 8.15 in the overlaying water to a minimum value of 7.3 at a depth of 1.2 mm. Further down in the sediment the pH increased to about 7.8 and remained constant. Most-probable-number (MPN) counts of chemolithoautotrophic sulfur-oxidizing bacteria revealed nearly constant numbers along the vertical profile; the cell concentration ranged from 0.93 x 10(5) to 9.3 x 10(5) cells per g of sediment. A specific PCR was used to detect the presence of Thiomicrospira cells in the MPN count preparations and to determine their 16S rRNA sequences. The concentration of Thiomicrospira cells did not decrease with depth. It was found that Thiomicrospira strains were not dominant sulfur-oxidizing bacteria in this habitat. Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments followed by hybridization analysis with a genus-specific oligonucleotide probe revealed the diversity of Thiomicrospira strains in the MPN cultures. Sequence analysis of the highest MPN dilutions in which the genus Thiomicrospira was detected revealed that there were four clusters of several closely related sequences. Only one of the 10 Thiomicrospira sequences retrieved was related to sequences of known isolates from the same habitat. Slot blot hybridization of rRNA isolated from different sediment layers showed that, in contrast to the concentration of Thiomicrospira cells, the concentration of Thiomicrospira-specific rRNA decreased rapidly in the region below the oxic layer of the sediment. This study revealed the enormous sequence diversity of closely related microorganisms present in one habitat, which so far has been found only by sequencing molecular isolates. In addition, it showed that most of the Thiomicrospira populations in the sediment studied were quiescent.
ABSTRACT
We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 &mgr;m during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10(8) SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.
