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

Phenotypic and genotypic analysis was carried out on four iron- and sulfur-oxidizing acidophilic bacteria (the "NO-37 group") isolated from different parts of the world. 16S rRNA phylogeny showed that they are highly related to each other, but are less related to the type strain of Acidithiobacillus ferrooxidans. The NO-37 group isolates are obligate chemolithoautotrophs, facultative anaerobes, diazotrophic, and psychrotolerant. They are less tolerant of extremely low pH, and in contrast to At. ferrooxidans (T), all of the NO-37 group isolates are motile. The GC contents of genomic DNA of the NO-37 group isolates were around 56 mol% and the DNA-DNA hybridization value between genomic DNA of isolate NO-37 and At. ferrooxidans (T) was 37%. It also appears that the bacteria of the NO-37 group have a different biochemical mechanism for oxidizing ferrous iron than At. ferrooxidans (T); the gene coding for the archetypal rusticyanin (RusA) was not detected in any of the NO-37 group isolates, rather a gene coding for a homologous protein (RusB) was amplified from three of the four novel isolates. Isolates of the NO-37 group clearly belong to a species that is different to those already recognized in the genus Acidithiobacillus, for which the name Acidithiobacillus ferrivorans is proposed.

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