The Tinto River (Huelva, southwestern Spain) is an extreme environment with a rather constant acidic pH along the entire river and a high concentration of heavy metals. The extreme conditions of the Tinto ecosystem are generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt. Molecular ecology techniques were used to analyze the diversity of this microbial community. The community's composition was studied by denaturing gradient gel electrophoresis (DGGE) using 16S rRNA and by 16S rRNA gene amplification. A good correlation between the two approaches was found. Comparative sequence analysis of DGGE bands showed the presence of organisms related to Leptospirillum spp., Acidithiobacillus ferrooxidans, Acidiphilium spp., "Ferrimicrobium acidiphilum," Ferroplasma acidiphilum, and Thermoplasma acidophilum. The different phylogenetic groups were quantified by fluorescent in situ hybridization with a set of rRNA-targeted oligonucleotide probes. More than 80% of the cells were affiliated with the domain Bacteria, with only a minor fraction corresponding to Archaea. Members of Leptospirillum ferrooxidans, Acidithiobacillus ferrooxidans, and Acidiphilium spp., all related to the iron cycle, accounted for most of the prokaryotic microorganisms detected. Different isolates of these microorganisms were obtained from the Tinto ecosystem, and their physiological properties were determined. Given the physicochemical characteristics of the habitat and the physiological properties and relative concentrations of the different prokaryotes found in the river, a model for the Tinto ecosystem based on the iron cycle is suggested.Microorganisms that are able to develop under extreme conditions have recently attracted considerable attention because of their peculiar physiology and ecology. These extremophiles also have important biotechnological applications (41,44,49,51). Acidic environments are especially interesting because, in general, the low pH of the habitat is the consequence of microbial metabolism (24) and not a condition imposed by the system as is the case in many other extreme environments (temperature, ionic strength, high pH, radiation, pressure, etc.). The Tinto River, a 100-km-long river in southwestern Spain, is an example of such an extreme acidic ecosystem; it has a low pH (between 1.5 and 3

The bacterial diversity assessed from clone libraries prepared from rRNA (two libraries) and ribosomal DNA (rDNA) (one library) from polychlorinated biphenyl (PCB)-polluted soil has been analyzed. A good correspondence of the community composition found in the two types of library was observed. Nearly 29% of the cloned sequences in the rDNA library were identical to sequences in the rRNA libraries. More than 60% of the total cloned sequence types analyzed were grouped in phylogenetic groups (a clone group with sequence similarity higher than 97% [98% for Burkholderia and Pseudomonas-type clones]) represented in both types of libraries. Some of those phylogenetic groups, mostly represented by a single (or pair) of cloned sequence type(s), were observed in only one of the types of library. An important difference between the libraries was the lack of clones representative of the Actinobacteria in the rDNA library. The PCB-polluted soil exhibited a high bacterial diversity which included representatives of two novel lineages. The apparent abundance of bacteria affiliated to the beta-subclass of the Proteobacteria, and to the genus Burkholderia in particular, was confirmed by fluorescence in situ hybridization analysis. The possible influence on apparent diversity of low template concentrations was assessed by dilution of the RNA template prior to amplification by reverse transcription-PCR. Although differences in the composition of the two rRNA libraries obtained from high and low RNA concentrations were observed, the main components of the bacterial community were represented in both libraries, and therefore their detection was not compromised by the lower concentrations of template used in this study.

A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain. Two morphotypes were abundant in these environments: rods and square or square-like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain-specific probes. FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square-like prokaryotes. These uncultured prokaryotes have the morphology of Walsby's square bacteria. Additionally, FISH with a probe targeted to the genus Haloarcula, members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community.

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4′,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of theCytophaga-Flavobacterium cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.

The community structure of sulfate-reducing bacteria (SRB) in an intertidal mud flat of the German Wadden Sea (Site Dangast, Jade Bay) was studied and related to sedimentary biogeochemical gradients and processes. Below the penetration depths of oxygen (~3 mm) and nitrate (~4 mm), the presence of dissolved iron and manganese and the absence of dissolved sulfide indicated suboxic conditions within the top 10 cm of the sediment. Moderate to high bacterial sulfate reduction rates were measured with radiotracers throughout the sediment, and dissimilatory sulfate reduction was also demonstrated by the presence of acid-volatile sulfides (AVS, essentially iron monosulfide). Stable sulfur isotope discrimination between dissolved sulfate and AVS was dominated by sulfate reduction. The diversity of SRB was studied using denaturant gradient gel electrophoresis of 16S rDNA, phospholipid fatty acid analysis and counting viable cells with the most probable number technique. Phylogenetic groups of SRB identified with these techniques were almost evenly distributed throughout the top 20 cm of the sediment. Application of fluorescence in situ hybridization, however, demonstrated a maximum of active members of the Desulfovibrio and Desulfosarcina-Desulfococcus-Desulfofrigus groups between 2 and 3 cm depth. These 2 groups encompass acetate and lactate utilizing SRB. The coincidence of this SRB maximum with a local maximum of sulfate reduction rates and the depletion of acetate and lactate reflects the microbiological processes related to sulfate reduction.