alt="Photos depict the comparison between ferruginous deposits of the Burns Formation (a) cropping up in the Endurance Crater and those formed by acidic fluvial activity (b) in Rio Tinto. In the fluvial deposits of Rio Tinto, jarosite and hematite were detected using different techniques such as X-ray diffraction (c), which form as a consequence of the low pH. "/>
Figure 2.2 Comparison between ferruginous deposits of the Burns Formation (a) cropping up in the Endurance Crater and those formed by acidic fluvial activity (b) in Rio Tinto. In the fluvial deposits of Rio Tinto, jarosite and hematite were detected using different techniques such as X-ray diffraction (c), which form as a consequence of the low pH. The same mineral association has been found on Mars. (Image credits: image A, PIA07110, NASA/JPL/Cornell; images B & C, the authors).
Considering the geomicrobiological characteristics of the Tinto ecosystem we propose that Rio Tinto is mainly under the control of iron [2.7]. Iron oxidizing microorganisms are responsible for the solubilization of the massive metal sulfides of the IPB and the associated high concentrations of iron and sulfate measured in the river. Iron has diverse properties of biological interest, which make the Rio Tinto basin an attractive astrobiological precedent [2.11]. Reduced iron is a good electron donor for respiration (both aerobic and anaerobic). Oxidized iron is a useful electron acceptor for anaerobic respiration. Oxidized iron is also a good buffer for controlling the pH of the system. Moreover, it has been shown that soluble ferric iron can protect sensitive organisms from damaging UV radiation [2.54] [2.55].
This iron-controlled environment appears to be adequate for the chemolithotrophic microorganisms detected in the Tinto basin. Nonetheless, taking into consideration that eukaryotic diversity seems to be much greater than prokaryotic [2.72] [2.5] [2.3], and that most of the primary production in the system depends on eukaryotic photosynthesis, how do eukaryotes benefit from adapting to an extreme acidic environment with high concentration of toxic heavy metals? One possible answer to this question might be related to free access to an indispensable element for life, iron. Iron is difficult to obtain at neutral pH, due to its meager solubility in this condition [2.14] [2.77] [2.19] [2.18]. A reliable advantage for the eukaryotes growing in the Tinto basin is an unrestricted iron supply provided by the chemolithotrophy promoted by the extremely high concentration of IPB iron sulfides [2.53] [2.7] [2.11].
2.6 The Iberian Pyrite Belt Dark Biosphere
As discussed above, we favor the hypothesis that the extreme conditions of pH and the high concentration of heavy metals detected along the Tinto basin is the result of an underground bioreactor in which interaction of metal sulfides, underground water and chemolithoautotrophic microorganisms generates the metabolic products, mainly iron and sulfuric acid, detected in the river. To demonstrate this hypothesis two devoted drilling projects to intersect this subsurface bioreactor and obtain information on the oxidation of metal sulfides in anaerobic conditions were carried out.
The central objective of the first, the Mars Astrobiology Research and Technology Experiment (MARTE project), a joint effort between the Centro de Astrobiología (CAB, CSIC-INTA) and the NASA Astrobiology Institute (NAI), was to gather information about the microbial activity operating in the subsurface of the IPB. Peña de Hierro (Iron Mountain, a recurrent name associated with mining operations), on the north flank of Rio Tinto anticline, was selected for drilling. Complex massive sulfide lenses or stockwork veins of pyrite and quartz generated by hydrothermal activity can be found at the upper part of the IPB volcanic sequence [2.69]. Faults intersect the Early Carboniferous volcanic tuff-hosted pyrite bodies.
Three boreholes, BH1, BH4 and BH8, were continuously cored by rotary diamond-bit drilling using a wireline system that produced 60 mm diameter cores, recovered inside a plastic liner to prevent excess contact between the core and the drilling fluid. Tap water with NaBr as a contamination tracer was used as drilling fluid to refrigerate the bit. Retrieved cores were placed in plastic bags, flushed with N2 to remove oxygen, sealed and transported to a nearby laboratory in the Museo Minero de Riotinto. Cores were then placed in an anaerobic chamber and samples were obtained aseptically from the interior of the selected cores with a modified hand drill operated at low speed (Figure 2.3).
Water from upslope springs was used to characterize the groundwater before contacting the ore body. The water from these springs had a neutral pH, a low ionic content and was saturated with O2. The environmental conditions within the ore body were obtained from the analysis of the core samples of two drilling boreholes, BH4 and BH8, at a depth of 165 mbs, separated by a distance of 10 m. The water table was detected at 90 mbs in both boreholes.
Wells were cased with PVC tubes to avoid collapse of the borehole walls and to install multilevel diffusion samplers (MLDS) at different depths for the analysis of ions and gases of the underground aquifer [2.40] [2.9]. Ionic chromatography analyses of the core samples leachates were used to evaluate the potential resources for microbial metabolisms and detect drilling contamination. Samples with Br concentrations above the background level were not analyzed further. Sulfate, a good indicator of the bio-oxidation of metal sulfides, was abundant in most samples. Both reduced and oxidized iron were extracted from several core samples evidencing an active iron cycle. Nitrate and nitrite were also detected in high concentrations in different samples [2.40] [2.9].
Figure 2.3 Processing the selected cores for the generation of samples in an anaerobic chamber in the Museo Minero laboratory. (Image credit: the authors).
Twice a year the MLDS were analyzed to follow the evolution of fluid formation in the boreholes. Similar patterns were observed for both boreholes. The average pH was ca. 3.5 and remained acidic for two years after drilling. Sulfate concentrations were lower than in rock leachates. The dissolved, oxidized and reduced iron ratios were variable along the length of the borehole, underlying the functional activity of the iron cycle. The highest concentration of dissolved H2 was found in the upper part of the water table and dissolved methane was detected in many samples, indicating methanogenic activity in the subsurface of the IPB [2.40] [2.9].
Core samples from both boreholes were examined with an antibody microarray (LDCHIP200) [2.85] and an oligonucleotide hybridization microarray [2.50], giving positive signals for sulfur and iron oxidizers, methanogenic archaea, sulfate reducers and Gram-positive bacteria. Denitrifying and hydrogenotrophic bacteria were identified by 16S rRNA cloning and sequencing. Enrichment cultures showed the presence of aerobic pyrite and iron oxidizers, anaerobic respiration of thiosulfate using nitrate, sulfate reducers and methanogens at different depths [2.87] [2.11].
The environment down-gradient from the metal sulfide ore body was sampled by drilling borehole BH1. Compared to BH4 and BH8 boreholes, BH1 showed lower iron and sulfate concentrations in the leachates while sulfate concentrations in the MLDS were much higher, indicating that the groundwater had a strong interaction with the ore. Dissolved hydrogen had lower concentrations and dissolved methane had higher concentration in BH1 than in BH4 and BH8. Enrichment cultures with samples from this borehole showed mainly methanogenic and sulfate-reducing activities [2.40] [2.9].
To further investigate the characteristics of the subsurface geomicrobiology of the IPB, researchers from the Centro de Astrobiología were granted an Advance ERC project, Iberian Pyrite Belt Subsurface Life Detection (IPBSL, 2011–2015) [2.10]. Two geophysical techniques, Time-domain Electromagnetic Sounding and Electric Resistivity Tomography, were used to obtain more precise information on subsurface areas most likely intersecting the underground bioreactor. Two
