Mónica Sánchez-Román

Aerobic microbial dolomite at the nanometer scale: Implications for the geologic record

Microbial experiments are the only proven approach to produce experimental dolomite under Earths surface conditions. Although microbial metabolisms are known to induce dolomite precipitation by favoring dolomite growth kinetics, the involvement of microbes in the dolomite nucleation process is poorly understood. In particular, the nucleation of microbially mediated dolomite remains a matter for investigation because the metabolic diversity involved in this process has not been fully explored. Herein we demonstrate that Halomonas meridiana and Virgibacillus marismortui, two moderately halophilic aerobic bacteria, mediate primary precipitation of dolomite at low temperatures (25, 35 °C). This report emphasizes the biomineralogical implications for dolomite formation at the nanometer scale. We describe nucleation of dolomite on nanoglobules in intimate association with the bacterial cell surface. A combination of both laboratory culture experiments and natural samples reveals that these nanoglobule structures may be: (1) the initial step for dolomite nucleation, (2) preserved in the geologic record, and (3) used as microbial tracers through time and/or as a proxy for ancient microbial dolomite, as well as other carbonate minerals.

Microbial nucleation of Mg-rich dolomite in exopolymeric substances under anoxic modern seawater salinity: New insight into an old enigma

Sulfate-reducing bacteria are known to mediate dolomite formation under hypersaline conditions, but details of the crystal nucleation process are still poorly constrained. Our laboratory study demonstrates for the first time that Desulfobulbus mediterraneus, a marine sulfate-reducing bacterium, mediates primary precipitation of Mg-rich dolomite under anoxic conditions in media replicating modern seawater chemistry at low temperature (21 °C). Precipitation of crystals was associated with extracellular polymeric substances in a monospecific biofilm, providing templates for nucleation by altering the molar Mg/Ca ratio. After initial nucleation of single nanospherulites (∼50 nm), growth was mediated by aggregation, resulting in spherulites of ∼2–3 μm in diameter. Nucleation led to differences in Mg/Ca ratios and δ44/40Ca values among the organic material (i.e., biofilm including cells and extracellular polymeric substances; 0.87 ± 0.01 [2 SD] and 0.48‰ ± 0.11‰ [2 SE], respectively), the crystals (1.02 ± 0.11 [2 SD] and <−0.08‰ ± 0.24‰ [2 SE], respectively), and the liquid bulk medium after mineral precipitation (4.53 ± 0.04 [2 SD] and 1.10‰ ± 0.24‰ [2 SE], respectively). These data indicate a two-step fractionation process involved in the sequestration of Ca from the solution into the crystal lattice of the mineral precipitated. Our results demonstrate the capability of extracellular polymeric substances to overcome kinetic inhibition, fostering the formation of kinetically less favorable Mg-rich dolomite, and they also question the applicability of the Ca isotopic system as a proxy for paleogeochemistry of seawater.

A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars.

The Atacama Desert has long been considered a good Mars analogue for testing instrumentation for planetary exploration, but very few data (if any) have been reported about the geomicrobiology of its salt-rich subsurface. We performed a Mars analogue drilling campaign next to the Salar Grande (Atacama, Chile) in July 2009, and several cores and powder samples from up to 5 m deep were analyzed in situ with LDChip300 (a Life Detector Chip containing 300 antibodies). Here, we show the discovery of a hypersaline subsurface microbial habitat associated with halite-, nitrate-, and perchlorate-containing salts at 2 m deep. LDChip300 detected bacteria, archaea, and other biological material (DNA, exopolysaccharides, some peptides) from the analysis of less than 0.5 g of ground core sample. The results were supported by oligonucleotide microarray hybridization in the field and finally confirmed by molecular phylogenetic analysis and direct visualization of microbial cells bound to halite crystals in the laboratory. Geochemical analyses revealed a habitat with abundant hygroscopic salts like halite (up to 260 g kg(-1)) and perchlorate (41.13 μg g(-1) maximum), which allow deliquescence events at low relative humidity. Thin liquid water films would permit microbes to proliferate by using detected organic acids like acetate (19.14 μg g(-1)) or formate (76.06 μg g(-1)) as electron donors, and sulfate (15875 μg g(-1)), nitrate (13490 μg g(-1)), or perchlorate as acceptors. Our results correlate with the discovery of similar hygroscopic salts and possible deliquescence processes on Mars, and open new search strategies for subsurface martian biota. The performance demonstrated by our LDChip300 validates this technology for planetary exploration, particularly for the search for life on Mars.

Amorphous Ca-phosphate precursors for Ca-carbonate biominerals mediated by Chromohalobacter marismortui

Although diverse microbial metabolisms are known to induce the precipitation of carbonate minerals, the mechanisms involved in the bacterial mediation, in particular nucleation, are still debated. The study of aragonite precipitation by Chromohalobacter marismortui during the early stages (3–7 days) of culture experiments, and its relation to bacterial metabolic pathways, shows that: (1) carbonate nucleation occurs after precipitation of an amorphous Ca phosphate precursor phase on bacterial cell surfaces and/or embedded in bacterial films; (2) precipitation of this precursor phase results from local high concentrations of PO43− and Ca2+ binding around bacterial cell envelopes; and (3) crystalline nanoparticles, a few hundred nanometres in diametre, form after dissolution of precursor phosphate globules, and later aggregate, allowing the accretion of aragonite bioliths.

Microbial Dolomite Precipitation under Aerobic Conditions: Results from Brejo do Espinho Lagoon (Brazil) and Culture Experiments

Microbially mediated high Mg-calcite and dolomite precipitation occurs under oxic conditions in Brejo do Espinho lagoon, Brazil, within the upper 5 cm below the sediment/water interface. With burial to < 25 cm in the sediment sequence, early diagenesis associated with sulfate reducing bacterial activity transforms the mixed carbonate mineralogy to 100% dolomite, as the pore water becomes undersaturated with respect to calcite, while remaining supersaturated with respect to dolomite. Laboratory culture experiments using moderately halophilic aerobic bacteria (Virgibacillus marismortui and Marinobacter sp.) isolated from the uppermost part of the microbial mat in Brejo do Espinho succinctly demonstrate that microbially mediated dolomite precipitation can occur under ambient Earth’s surface conditions in the presence of oxygen. These results add an additional metabolic process, aerobic respiration, to bacterial sulfate reduction and methanogenesis, which have previously been identified with dolomite formation. Furthermore, the formation of carbonate minerals with spherulitic structures in both the natural environment and laboratory culture experiments points to microbial involvement, as recognized in numerous other modern environments and ancient systems. This study suggests that previously recognized modern dolomiteforming environments, such as the supratidal areas of Andros Island, Bahamas with Recent dolomite crusts, should be revisited to evaluate the importance of aerobic respiration in dolomite precipitation.

Microbial mediated formation of Fe-carbonate minerals under extreme acidic conditions

Discovery of Fe-carbonate precipitation in Rio Tinto, a shallow river with very acidic waters, situated in Huelva, South-western Spain, adds a new dimension to our understanding of carbonate formation. Sediment samples from this low-pH system indicate that carbonates are formed in physico-chemical conditions ranging from acid to neutral pH. Evidence for microbial mediation is observed in secondary electron images (Fig. 1), which reveal rod-shaped bacteria embedded in the surface of siderite nanocrystals. The formation of carbonates in Rio Tinto is related to the microbial reduction of ferric iron coupled to the oxidation of organic compounds. Herein, we demonstrate for the first time, that Acidiphilium sp. PM, an iron-reducing bacterium isolated from Rio Tinto, mediates the precipitation of siderite (FeCO3) under acidic conditions and at a low temperature (30°C). We describe nucleation of siderite on nanoglobules in intimate association with the bacteria cell surface. This study has major implications for understanding carbonate formation on the ancient Earth or extraterrestrial planets.

Specific jarosite biomineralization by Purpureocillium lilacinum, an acidophilic fungi isolated from Río Tinto

Río Tinto (Huelva, southwestern Spain) is an extreme environment with a remarkably constant acidic pH and a high concentration of heavy metals, conditions generated by the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyrite Belt (IPB). Fungal strains isolated from the Tinto basin were characterized morphologically and phylogenetically. The strain identified as Purpureocillium lilacinum specifically induced the formation of a yellow-ocher precipitate, identified as hydronium-jarosite, an iron sulfate mineral which appears in abundance on the banks of Río Tinto. The biomineral was characterized by X-ray diffraction (XRD) and its formation was observed with high-resolution transmission electron microscopy (TEM) and scanning electron microscopy (SEM) coupled to energy-dispersive X-ray spectroscopy (EDX) microanalysis. Jarosite began to nucleate on the fungal cell wall, associated to the EPS, due to a local increase in the Fe(3+) /Fe(2+) ratio which generated supersaturation. Its formation has been also observed in non-viable cells, although with much less efficiency. The occurrence of P. lilacinum in an ecosystem with high concentrations of ferric iron and sulfates such as Río Tinto suggests that it could participate in the process of jarosite precipitation, helping to shape and control the geochemical properties of this environment.

Prokaryotic communities and operating metabolisms in the surface and the permafrost of Deception Island (Antarctica)

In this study we examined the microbial community composition and operating metabolisms on the surface and in the permafrost of Deception Island, (Antarctica) with an on site antibody microarray biosensor. Samples (down to a depth of 4.2 m) were analysed with LDChip300 (Life Detector Chip), an immunosensor containing more than 300 antibodies targeted to bacterial and archaeal antigens. The immunograms showed positive antigen-antibody reactions in all surface samples (lichens, pyroclasts) and the top layer of the permafrost. The results indicated the presence of exopolysaccharides, bacteria belonging to the Alpha-, Delta- and Gammaproteobacteria, Bacteroidetes, Gram-positive Actinobacteria and Firmicutes, as well as archaeal species, most probably Methanobacterium spp. Positive reactions with antibodies to proteins and peptides revealed the presence of nitrogen fixation (NifHD, GlnB, HscA), methanogenic (McrB), iron homeostasis and iron scavenging (ferritins and DPS proteins) proteins, as well as ABC transporters, which indicated that these processes were operating at the time of sampling. These results were validated with other molecular ecology techniques such as oligonucleotide microarrays, 16S bacterial rRNA gene sequence analysis, aerobic viable counts and microscopy. Molecular ecology results showed a differentiated pattern along the depth of the drill, being the top active layer the most diverse, with Acidobacteria, Actinobacteria, Proteobacteria, Bacteroidetes and the phototrophs Cyanobacteria and Chloroflexi as dominant groups. Actinobacteria and Firmicutes were dominant in depths from 0.5 to 2 m, and Betaproteobacteria from 3 to 4.2 m. The geochemical analysis revealed the presence of low molecular weight organic acids (acetate, formate) which could be used by microorganisms as energy sources for sulfate, nitrate and metal reduction under anaerobic conditions.

Iberian Pyrite Belt Subsurface Life (IPBSL), a Drilling Project of Biohydrometallurgical Interest

The geomicrobiological characterization of Río Tinto, an extreme acidic environment, has proven the importance of the iron cycle, not only in generating the extreme conditions of the habitat (low pH, high concentration of toxic heavy metals) but also in maintaining the high level of microbial diversity detected in the water column and the sediments. The extreme conditions detected in the Tinto basin are not the product of industrial contamination but the consequence of the presence of an underground bioreactor that obtains its energy from the massive sulfide minerals of the Iberian Pyrite Belt (IPB). To test this hypothesis, a drilling project (IPBSL) to intersect ground waters interacting with the mineral ore is under way, to provide evidence of subsurface microbial activities. A dedicated geophysical characterization of the area selected two drilling sites due to the possible existence of water with high ionic content. Two wells have been drilled in Peña de Hierro, BH11 and BH10, with depths of 340 and 630 meters respectively, with recovery of cores and generation of samples in anaerobic and sterile conditions. The geological analysis of the retrieved cores showed an important alteration of mineral structures associated with the presence of water, with production of expected products from the bacterial oxidation of pyrite. Ion chromatography of water soluble compounds from uncontaminated samples showed the existence of putative electron donors, electron acceptors, as well as variable concentration of metabolic organic acids, which suggest the presence of an active subsurface ecosystem associated to the high sulfidic mineral content of the IPB. Enrichment cultures from selected samples showed evidences of an active iron and sulfur cycle, together with unexpected methanogenic, methanotrophic and acetogenic activities. The geological, geomicrobiological and molecular biology analyses which are under way, should allow the characterization of this ecosystem of biohydrometallurgical interest

Nucleation of Fe-rich phosphates and carbonates on microbial cells and exopolymeric substances

Although phosphate and carbonate are important constituents in ancient and modern environments, it is not yet clear their biogeochemical relationships and their mechanisms of formation. Microbially mediated carbonate formation has been widely studied whereas little is known about the formation of phosphate minerals. Here we report that a new bacterial strain, Tessarococcus lapidicaptus, isolated from the subsurface of Rio Tinto basin (Huelva, SW Spain), is capable of precipitating Fe-rich phosphate and carbonate minerals. We observed morphological differences between phosphate and carbonate, which may help us to recognize these minerals in terrestrial and extraterrestrial environments. Finally, considering the scarcity and the unequal distribution and preservation patterns of phosphate and carbonates, respectively, in the geological record and the biomineralization process that produces those minerals, we propose a hypothesis for the lack of Fe-phosphates in natural environments and ancient rocks.