(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 [2.74]. A comparative analysis of the microbial diversity associated with sediments and the correspondent water column showed a significantly higher level of diversity in the sediments [2.49]. Most of the microorganisms identified in this study were somehow related to the iron cycle. Most of them have been isolated or identified in biomining operations or AMD sites [2.60] [2.61] [2.57] [2.58] [2.92] [2.47]. Nonetheless, some bacteria, such as members of Firmicutes, Acidobacteria, Actinobacteria, Planctomycetes and Chloroflexi phyla, were identified for the first time in the Tinto basin [2.49].
A thorough analysis of two closely located anoxic sediments from the origin of the river has recently shown major phylogenetic differences. In one of the sediments, members of the Firmicutes followed by Acidobacteria phyla corresponded to the most numerous group of Bacteria. In the other sediment, members of the Proteobacteria followed by members of the Actinobacteria phyla were the most represented [2.94]. Members of Firmicutes and Acidobacteria were also present but at rather low numbers. Sulfate-reducing bacteria have always been identified in the strict anoxic conditions existing in the lower part of the sediments, indicating that this energy conservation system is a frequent metabolic activity in the anaerobic part of the ecosystem, probably due to the high concentration of sulfates detected [2.75] [2.94] [2.11].
2.5 Tinto Basin Sedimentary Geomicrobiology
As previously mentioned, most of the biomass from the Tinto ecosystem forms colorful biofilms on the riverbed and on its rock surfaces. Iron minerals precipitate on the surface of these biofilms according to the hydrological cycles, consolidating into distinct fluvial terraces [2.35] [2.37].
During the dry season the evaporation of river water drives precipitation of schwertmannite and hydronium jarosite, whereas gypsum, coquimbite, copiapite and other sulfate minerals are transported to the surface by capillary action, generating effluorescences [2.37]. During the rainy season, hydrolysis of sulfates in addition to the iron hydrolysis results in the precipitation of amorphous iron oxyhydroxides and nanophase goethite. In the oldest river terraces, increasing goethite crystallinity and its replacement by hematite can be observed (Figure 2.2).
Biosignatures, such as textures of coated microbial streamers, surface blisters generated by biogenic gas and microfossils preserved in iron oxides, can be detected in the sedimentary rocks, which could be of help in designing strategies for future astrobiological exploration missions [2.37] [2.39] [2.8]. Until recently, the extreme conditions of pH and high concentration of toxic heavy metals identified in the Tinto basin were considered the consequence of over 5000 years of mining activities [2.51] [2.68] [2.27]. However, recent geological, geophysical and hydrogeological information does not support this assumption. The recharge area of Peña de Hierro aquifer was detected northwest of the pit lake at a depth ranging from 100 to 400 mbs [2.56]. Its groundwater moves southwards along the fracture network and when it reaches the massive sulfide bodies it activates the metabolism of chemolithotrophic microorganisms, generating acidic waters with high concentrations of iron and sulfate [2.38] [2.56]. This acidic groundwater is pumped along open strike-slip normal faults and eventually reaches the surface, where it sources the acidic springs that feed the Rio Tinto headwaters. Thus, the acidic waters with high content of iron and sulfate are the consequence of the subsurface oxidation of metal sulfides, mainly pyrite, by chemolithotrophic microorganisms operating in strict anaerobic conditions. Consequently, mining activities that brought sulfidic minerals to the surface are not the main cause of the distinctive acidity and high concentration of heavy metals detected in the river. This hypothesis is strongly supported by the sedimentary record of the ancient terraces deposited along the Tinto basin [2.37]. The oldest, containing finely laminated and massive ironstones, has been dated 2.1 (million years ago) Ma. This means that several millions of years before the oldest recorded mining activity, the natural bioleaching of native unexposed sulfidic minerals (subterranean and, as a consequence, in anoxic conditions) gave rise to the ionic conditions that generated the old terraces [2.38]. Furthermore, the oldest gossan deposits in the Tinto basin, representing the remaining materials of the in-situ bioleaching of the sulfidic deposits, were dated older than 7 Ma [2.32], underscoring the natural origin of the river.
Recently, the remarkable biomineralization of hydronium jarosite in non-permissive ionic conditions by Purpureocillium lilacinum, a filamentous fungi isolated from Rio Tinto [2.82], has been described. In addition, siderite was identified in the modern sediments of the river [2.42], which was also produced in cultures of the acidophilic iron reducer Acidiphilium sp., one of the major microorganisms identified in the Tinto basin [2.97]. These results clearly show that the presence of microorganisms can generate nucleation sites which could radically modify the expected mineral precipitation schemes predicted by the global physicochemical conditions of the environment in which they develop [2.82] [2.83] [2.84]. Furthermore, the detection of organic molecules in the oldest Rio Tinto terraces indicates that even in the extreme acidic and oxidant conditions of the system this ferruginous environment can preserve the molecular information of organisms that have inhabited this extreme environment for long periods of time [2.22] [2.89].
