Microbial ecology of an extreme acidic environment, the Tinto River

Abstract

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.

FIG. 1.

Tinto River location, showing the sampling sites used in this work.

FIG. 2.

DGGE fingerprints of 16S rRNA and reverse-transcribed 16S rRNA (marked by asterisks) obtained by using universal primers for members of the domain Bacteria. (A) Samples from June 1999; (B) samples from October 1999. Lane numbers correspond to sampling sites. Arrows label different bands.

FIG. 3.

Phylogenetic affiliations of DGGE sequences with strong similarities to Acidithiobacillus spp. The tree is based in a parsimony analysis that includes only complete or almost-complete sequences of representative bacteria (38). The topology of the tree resulted from insertion of the partial DGGE sequences without modifying its topology during sequence positioning (ARB software [http://www.mikro.biologie.tu-muenchen.de]). Acidithiobacillus ferrooxidans sequences grouped into four clusters: a, b, c, and d. Sequences retrieved from DGGE bands are designated by the letter B followed by the number of the band. The specificities of the four probes designed to identify each group are shown.

FIG. 4.

Phylogenetic affiliations of DGGE sequences with strong similarities to Leptospirillum spp. The tree was constructed as described in the legend to Fig. 3. Leptospirillum spp. grouped into three clusters: a, b (L. ferriphilum), and c (L. ferrooxidans). Sequences from group c are distributed into two subgroups named c₁ and c₂. All the DGGE sequences from the Tinto River fall into group c. The specificities of the probes (LEP634, LEP154, LEP636, LEP439, and LEP432) designed to identify members of the different groups are shown. The specificity of probe NTR712 (specific for members of the Nitrospira phylum with the exception of group a of leptospirilli) is also shown. ^a, Groups defined by Bond et al. (6); ^b, species defined by Coram and Rawlings (12).

FIG. 5.

Epifluorescence micrographs of bacteria from the Tinto River. (A, C, and E) DAPI-stained cells in RT9, RT1, and RT10 samples, respectively; (B, D, and F) same fields as A, C, and E, respectively, showing cells hybridized with different specific probes. Samples were hybridized with probe ACT465c (Cy3 labeled), specific for group c of Acidithiobacillus ferrooxidans (B), probe ACD638 (Cy3 labeled), specific for Acidiphilium spp. (D), or probe LEP636 (Cy3 labeled), specific for group c of leptospirilli (F). Bars, 5 μm.

FIG. 6.

Geomicrobiological model of the Tinto River showing the roles of the different microorganisms identified in the ecosystem. Microorganisms are shown associated with their roles in the iron and sulfur cycles. The type size of the organism designation is proportional to the respective cell density.