significantly in their characteristics and as a consequence in their microbial ecology. High temperatures generated by biological activity facilitate the development of thermophilic and thermotolerant acidophiles. Acidic eco-systems associated with metal mining activities are, at the geological scale, rather young. Nonetheless, some mining activities, such as those in the Tinto area, which have been in exploitation for more than 5.000 years, have a somewhat relatively long history [2.68].
2.3 Rio Tinto Basin
Size (92 km long), pH (mean pH 2.3), high concentration of heavy metals (Fe, Cu, Zn, As…) and a high level of microbial diversity, mainly eukaryotic, make Rio Tinto an uncommon extreme acidic environment [2.72] [2.5] [2.6] [2.57] [2.2] [2.3] [2.13] (Figure 2.1). Rio Tinto rises in Peña de Hierro, in the core of the Iberian Pyrite Belt (IPB) and merges with the Atlantic Ocean at Huelva. The IPB is a geological unit 250 km long and 60 km wide located in the northernmost zone of the Variscan Iberian Massif. The IPB is one of the largest sulfidic deposits on Earth. Massive bodies of pyrite and chalcopyrite, as well as minor quantities of lead and zinc sulfides, constitute the main mineral ores of the IPB [2.17] [2.69]. Hydrothermalism during the Hercynian orogenesis was responsible for its generation [2.17] [2.70]. In the 1700 km2 river basin three zones can be clearly defined according to their chemical and geological characteristics: the northern (from Peña de Hierro to Niebla), the transitional (from Niebla to San Juan del Puerto) and the estuarine (from San Juan to the Atlantic Ocean). The last zone can be considered to have the most extreme conditions detected in the Tinto basin, because the microorganisms thriving in this part of the river have to face drastic changes in pH, from around 3 to neutrality, as a consequence of the twice daily tidal influence of the Atlantic Ocean [2.103]. Climographs exhibit characteristic basin bimodality, with humid and temperate periods (fall, winter and early spring), alternating with dry and warm periods (late spring and summer).
Figure 2.1 View of colorful filamentous algae in the red waters of the origin of Rio Tinto. (Image credit: the authors).
Oxygen content varies from saturation to strict anaerobic conditions, which agree with the range, -280 to +650 mV of redox potentials measured. The comparison with other local rivers indicates that the acidity and the concentration of iron in the Tinto basin are at least one order of magnitude higher than the acidic Odiel and Agrio rivers, both also associated with mining activities [2.72].
A peculiar characteristic of the Tinto ecosystem is its rather constant pH, which is the consequence of the buffer capacity of ferric iron.
(2.1)
When the river is diluted by neutral tributaries or rain, hydrolysis of ferric iron occurs, precipitating ferric hydroxides and generating protons. In the summer, intense heat evaporates the water in the river and protons are consumed, dissolving the ferric hydroxide precipitates. Due to this buffering capacity a pH of around 2.3 remains constant along the river course with the exception of the estuarine zone. Its dimensions and relatively easy access make the Tinto basin an excellent model for the study of microbial ecology associated with an extreme acidic environment [2.12].
2.4 Biodiversity in the Tinto Basin
Combining conventional and molecular microbial ecology methods allowed the most characteristic organisms associated with the Tinto basin to be identified [2.72] [2.57] [2.6] [2.49] [2.94] [2.96] [2.3]. Remarkably, over eighty per cent of the water column diversity corresponds to microorganisms belonging to only three bacterial genera: Leptospirillum spp. (strict aerobic iron oxidizers), Acidiphilium spp. (iron reducers), and Acidithiobacillus ferrooxidans (an aerobic iron oxidizer and an anaerobic iron reducer), all of them well-known members of the iron cycle [2.57].
Although other bacterial and archaeal iron-oxidizers (members of Ferrimicrobium, “Ferrovum,” Ferrimicrobium and Thermoplasma genera) or bacterial iron-reducers (members of Ferrimicrobium, Metallibacterium and Acidobacterium genera) have been identified in the Tinto ecosystem [2.57] [2.47] [2.49]. Their low numbers, as detected by fluorescence in situ hybridization, suggest that they have a less important role in the water column.
Regarding the sulfur cycle, only At. ferrooxidans was detected in significant numbers in the water column. Sulfate-reducing microorganisms were also detected in the sediments at different locations along the river [2.75] [2.49] [2.94] [2.95] [2.96] [2.44] [2.45], thus a subsidiary sulfur cycle is also operative along the course of the river.
It is assumed that the toxicity of high concentrations of heavy metals existing in extreme acidic environments impairs the development of eukaryotic microorganisms in these ecosystems [2.59]. Nevertheless, multicolored algal biofilms can be easily observed covering important areas along the river (Figure 2.1). A careful estimation of this phenomenon allowed López-Archilla and collaborators to conclude that more than sixty per cent of the primary production in the river is due to photosynthetic eukaryotic organisms [2.72]. Members of the Chlamydomonas, Chlorella and Euglena genera, all belonging to the Chlorophyta phylum, are the dominant eukaryotic microorganisms detected in the Tinto basin [2.5] [2.6] [2.2] [2.3]. Also, filamentous algae of the Klebsormidium and Zygnemopsis genera were identified in the river. Species of the Dunaliella and Cyanidium genera can be found colonizing the most extreme area in the origin of the river. Members of these genera are well known for their tolerance to toxic heavy metals [2.1] [2.3]. Large brown biofilms made of pennate diatoms of the genus Pinnularia can be detected along the course of the Tinto basin [2.2] [2.3] [2.4].
Unicellular and filamentous fungi are the most diverse eukaryotes detected in the Tinto basin [2.72] [2.73]. A recent characterization of Rio Tinto fungal diversity from samples obtained along the river has rendered three hundred and fifty isolates, which is a small representation of the fungal diversity existing in the Tinto basin. Internal transcribed spacer (ITS) sequence analysis showed that Ascomycetes is the most abundant phylum, followed by Basidiomycetes and Zygomycetes. Among the Ascomycetes, more than fifty per cent cluster within the Eurotiomycetes class, followed by Dothideomycetes, Sordariomycetes, Helotiales and Mucorales. A systematic survey of fungal metal tolerance showed that members of the Sordariomycetes and the Eurotiomycetes classes are the most resistant to high concentration of toxic heavy metals, in some cases several orders of magnitude higher than the concentration measured in the water column. Interestingly enough, many different fungal isolates from the Tinto basin can grow in the presence of high concentrations of individual toxic metals, such as As, Ni, Co, Cr, Cu, but their tolerance decreases severely in the presence of combinations of these toxic heavy metals. In addition, some metals with low toxicological profile, such as Fe and Al, can increase the fungal tolerance of individual or combined toxic metals.
Heterotrophic protists are also found along the river. The mixotrophic community is dominated by cercomonads and stramenopiles belonging to the Ochromonas, Bodo and Cercomonas genera. The protistan consumer community is characterized by two different ciliates identified as members of the Euplotes and Oxytricha genera. Amoebas related to the Vahlkampfia and Naegleria genera are found in the most acidic part of the river. Members of the heliozoan belonging to the Actinophyros genus are the most characteristic predators of the benthic food chain [2.5] [2.2] [2.3]. The bdelloid rotifer of the genus Rotifera is the only member of the animal phylum detected so far in Rio Tinto [2.5].
Due to the interest of aerobic chemolithotrophs in biohydrometallurgical processes, the characterization of the anoxic sediments from acidic environments had been neglected in the past, with few exceptions