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,
2.7 Methanogenesis in Non-Methanogenic Conditions
Although methane can be produced abiotically, more than eighty per cent of the methane existing in the Earth atmosphere is generated by methanogenic archaea. With only a few exceptions, methanogenic activities are normally detected in environments with circumneutral pH and negative (reduced) redox potentials [2.66] [2.102]. These conditions are very different from those detected in the Tinto basin (acidic and high (oxidized) redox potentials). For a long time, methanogenic activities were not considered to be operating in the Tinto basin for this reason.
After the detection of methane in the borehole fluids of the MARTE project and in the Martian atmosphere [2.46] [2.81], regular inspections for methanogenic activity were implemented in the study of the anoxic sediment of the Tinto basin. The first sampling station in which methane generation was detected was Campo de Galdérias, at the origin of the river [2.100]. Sediments from this station exhibited specific locations with negative reduced redox potential, surrounded by the high positive oxidation redox potential characteristic of the river. Pressure applied to the ground around this sampling site released gases occluded in the sediments. Microcosms using these reduced sediments showed very active methane generation after reaching negative redox potentials and a significant pH increase, following the spike with different methanogenic substrates. The highest methane production was obtained after addition of methanol [2.100].
A second site, JL Dam, was selected for further analysis. Cores from this sampling site exhibited distinctive black bands with negative reduced redox potential and circumneutral pH among acidic reddish-brown sediments with positive oxidized redox potentials, similar to those detected in the sediments collected along the course of the river. The sequence of amplified 16S rRNA gene from the blackish bands corresponded to Methanosaeta concilii. Enrichment cultures using different methanogenic substrates allowed the identification of M. concilii using organic substrates, Methanobacterium bryantii using H2 and Methanosarcina barkeri using methanol [2.100].
How can we explain the development of methanogenic activities in an ecosystem in which the characteristic pH and redox potential are the opposite of the required conditions? This apparent contradiction is resolved when we analyze the results at the microscopic level. The generation of micro-niches in a semi-solid matrix, such as sediments, or even more, in a solid matrix within a deep subsurface rock, could facilitate the growth of microorganisms generating environmental conditions quite different from the restrictive ones existing in the ecosystem [2.31]. As mentioned above, the use of fluorescence in situ hybridization allowed the detection of these micro-niches in the porous rocks of the deep subsurface of the IPB, many of which included biofilms with different types of microorganisms, sharing space and metabolisms [2.31].
The presence of methanogens in an environment controlled by iron has important astrobiological implications since it could be a model for the biotic generation of Martian atmospheric methane [2.46] [2.81] [2.105]. The recurrent argument that the environmental conditions of Mars are not suitable for generating biological methane could be contradicted by the methanogenic activities detected in the sediments of Rio Tinto and the subsurface of the IPB, a geochemical and mineralogical terrestrial analogue
