Читать книгу EXTREMOPHILES as Astrobiological Models - Группа авторов - Страница 39

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 boreholes, BH10 (620 mbs) and BH11 (340 mbs) were selected for drilling [2.56] (Figure 2.4).

Drilling was performed in similar conditions to those described previously for the MARTE project. Rock leachates obtained from samples at regular intervals were analyzed overnight by ion chromatography to determine the concentration of water-soluble anions, facilitating the selection of cores for further analysis using complementary methodologies. Chromatograms showed the presence of oxidized inorganic in ions, such as nitrate and sulfate, as well as reduced organic acids such as acetate. Proteins and sugars were also detected in different samples, demonstrating the existence of microorganisms at different depths.

Cores were logged at the drilling site and samples from selected cores were taken for further petrographic, mineralogical (XRD), elemental (ICP-MS) and stable isotopic analysis. Pyrite and its alteration products, hematite and magnetite, were identified mineralogically in samples from both boreholes. Iron and other metals were identified in leachates from these samples.

Gas chromatography of core samples from both boreholes detected H₂, CO₂ and CH₄. Samples from the BH10 borehole were analyzed with the immunosensor LDChip300, a new generation of antibody microarray containing three hundred antibodies with diverse and complementary specificity. Positive immunological reactions were detected using specific antibodies against sulfate-reducing bacteria and methanogenic archaea, which agree with the results obtained using other techniques.

Figure 2.4 Borehole BH11 drilling in Peña de Hierro. (Image credit: the authors).

DNA and RNA were efficiently extracted from core samples from both boreholes and most of them gave positive amplifications for bacterial 16S rRNA genes, which are currently under analysis by cloning and pyrosequencing. Samples from the two boreholes were fixed and stained for examination using fluorescence in situ hybridization (CARD-FISH). So far, the results show positive hybridization signals for both Bacteria and Archaea [2.31] and some specific phyla probes, including α-, β-and γ-Proteobacteria, and Gram-positive Bacteroidetes and Euryarchaea, at different depths (Figure 2.5). Further hybridizations with probes selected or designed after identification by sequence analysis are under development. Biofilms were detected in core samples using fluorescence in situ hybridization and sugar specific fluorescent probes [2.31]. This is an interesting observation because these results challenge the concept that under strict oligotrophic conditions, like those existing in the deep subsurface, microorganisms cannot afford to waste energy producing biofilms. This means that subsurface lifestyle in a solid matrix makes use of biofilms not only to control the bio-reactions in the micro-niches but also to efficiently interconnect them [2.31]. Different electron donors and acceptors have been used to prepare anaerobic enrichment cultures. The following activities have been detected using samples from both boreholes: iron and sulfur oxidizers, iron and sulfate reducers, methanogens, acetogens, methanotrophs and denitrifiers.

Figure 2.5 Bacteria detected using the CARD-FISH probe EUB338-1 at 420 mbs. (Image credit: the authors).

The results obtained so far in the MARTE and IPBSL drilling projects give us the following scenario: as groundwater encounters the volcanogenic-hosted massive sulfide (VHMS) system, both biotic and abiotic processes are triggered. Electron donors available for microbial metabolism include metal sulfides, ferrous iron, , hydrogen, nitrite and reduced organic matter. The list of possible electron acceptors includes ferric iron, sulfate, nitrate and CO₂. These compounds can support different anaerobic respiration metabolisms. This is not a complete list; further analysis of the enrichment cultures allowed us to isolate microorganisms developing in the strict anaerobic conditions detected in the subsurface [2.88] [2.67] and their physiological and genomic characterization should provide new insight into the array of metabolisms operating in the subsurface of the IPB. These results confirm the hypothesis that microorganisms are active in the subsurface of the IPB and are responsible for the extreme conditions detected in the river. This observation has important astrobiological implications [2.11] [2.43].