mechanism, producing, in this case, elemental sulfur as final product [2.99], and requiring the additional activity of sulfur oxidizing microorganisms to generate sulfuric acid. The main players in these reactions are the iron-oxidizing microorganisms responsible for maintaining a high concentration of the chemical oxidizing agent, ferric iron.
The acidophilic strict chemolithoautotroph Acidithiobacillus ferrooxidans (formerly known as Thiobacillus ferrooxidans) was isolated for the first time in a coal mine in the middle of the last century [2.23]. Although At. ferrooxidans can obtain energy by oxidizing both reduced sulfur and iron, bioenergetic considerations ignored the role of reduced iron as an important source of energy for chemolithotrophic organisms for many years [2.90] [2.7] [2.30]. But the isolation and characterization of the strict chemolithotroph Leptospirillum ferrooxidans, which can only grow using ferrous iron as its source of energy, and the evaluation of its important role in biohydrometallurgical operations, has finally changed this point of view [2.91] [2.61] [2.48] [2.30]. In addition, it is now well-known that iron can also be oxidized in anaerobic conditions through anoxygenic photosynthesis using reduced iron as reducing power [2.106] or anaerobic respirations using nitrate as an electron acceptor [2.16], although the mechanism in this case is still very controversial [2.64] [2.20] [2.65] [2.110].
The current demonstration that subsurface chemolithotrophic microorganisms participate very actively in the dark biosphere, already predicted by Darwin almost two hundred years ago, has opened interesting perspectives not only in microbial ecology but also in astrobiology [2.52] [2.15] [2.86] [2.21] [2.111] [2.9] [2.10] [2.12] [2.87] [2.43]. There is an increasing list of alternative sources of chemolithotrophic energy (H2,
Extreme acidic environments differ 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
