Antonio García-Moyano

Nematoda from the terrestrial deep subsurface of South Africa

Since its discovery over two decades ago, the deep subsurface biosphere has been considered to be the realm of single-cell organisms, extending over three kilometres into the Earth’s crust and comprising a significant fraction of the global biosphere. The constraints of temperature, energy, dioxygen and space seemed to preclude the possibility of more-complex, multicellular organisms from surviving at these depths. Here we report species of the phylum Nematoda that have been detected in or recovered from 0.9–3.6-kilometre-deep fracture water in the deep mines of South Africa but have not been detected in the mining water. These subsurface nematodes, including a new species, Halicephalobus mephisto, tolerate high temperature, reproduce asexually and preferentially feed upon subsurface bacteria. Carbon-14 data indicate that the fracture water in which the nematodes reside is 3,000–12,000-year-old palaeometeoric water. Our data suggest that nematodes should be found in other deep hypoxic settings where temperature permits, and that they may control the microbial population density by grazing on fracture surface biofilm patches. Our results expand the known metazoan biosphere and demonstrate that deep ecosystems are more complex than previously accepted. The discovery of multicellular life in the deep subsurface of the Earth also has important implications for the search for subsurface life on other planets in our Solar System.

Comparative microbial ecology study of the sediments and the water column of the Río Tinto, an extreme acidic environment

Due to its highly metalliferous waters and low pH, the Rio Tinto has shown its potential for modelling both acid mine drainage systems and biohydrometallurgical operations. Most geomicrobiological studies of these systems have focused on the oxic water column. A sequence-based approach in combination with in situ detection techniques enabled us to examine the composition and structure of the microbial communities associated with the suboxic and anoxic sediments along the river course and to compare them with the planktonic communities inhabiting the water column. The results obtained with the different approaches were consistent and revealed some major patterns: higher cell density and higher richness (75 vs. 48 operational taxonomic units) in the sediments than in the water column. The microbial communities were related but the river sediments appear to be enriched in certain populations, some of which have not previously been reported in the Rio Tinto basin. The differences detected between sampling stations along the river correlate with certain environmental parameters (e.g. iron concentration gradient). The biological and geochemical data show the importance of the sediments as representing a phase of particular high diversity, probably related to key metabolic processes within both the iron and the sulfur cycles.

An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA

An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.

Deciphering the Prokaryotic Community and Metabolisms in South African Deep-Mine Biofilms through Antibody Microarrays and Graph Theory

In the South African deep mines, a variety of biofilms growing in mine corridor walls as water seeps from intersections or from fractures represents excellent proxies for deep-subsurface environments. However, they may be greatly affected by the oxygen inputs through the galleries of mining activities. As a consequence, the interaction between the anaerobic water coming out from the walls with the oxygen inputs creates new conditions that support rich microbial communities. The inherent difficulties for sampling these delicate habitats, together with transport and storage conditions may alter the community features and composition. Therefore, the development of in situ monitoring methods would be desirable for quick evaluation of the microbial community. In this work, we report the usefulness of an antibody-microarray (EMChip66) immunoassay for a quick check of the microbial diversity of biofilms located at 1.3 km below surface within the Beatrix deep gold mine (South Africa). In addition, a deconvolution method, previously described and used for environmental monitoring, based on graph theory and applied on antibody cross-reactivity was used to interpret the immunoassay results. The results were corroborated and further expanded by 16S rRNA gene sequencing analysis. Both culture-independent techniques coincided in detecting features related to aerobic sulfur-oxidizers, aerobic chemoorganotrophic Alphaproteobacteria and metanotrophic Gammaproteobacteria. 16S rRNA gene sequencing detected phylotypes related to nitrate-reducers and anaerobic sulfur-oxidizers, whereas the EMChip66 detected immunological features from methanogens and sulfate-reducers. The results reveal a diverse microbial community with syntrophic metabolisms both anaerobic (fermentation, methanogenesis, sulphate and nitrate reduction) and aerobic (methanotrophy, sulphur oxidation). The presence of oxygen-scavenging microbes might indicate that the system is modified by the artificial oxygen inputs from the mine galleries.

Novel and Unexpected Microbial Diversity in Acid Mine Drainage in Svalbard (78° N), Revealed by Culture-Independent Approaches

Svalbard, situated in the high Arctic, is an important past and present coal mining area. Dozens of abandoned waste rock piles can be found in the proximity of Longyearbyen. This environment offers a unique opportunity for studying the biological control over the weathering of sulphide rocks at low temperatures. Although the extension and impact of acid mine drainage (AMD) in this area is known, the native microbial communities involved in this process are still scarcely studied and uncharacterized. Several abandoned mining areas were explored in the search for active AMD and a culture-independent approach was applied with samples from two different runoffs for the identification and quantification of the native microbial communities. The results obtained revealed two distinct microbial communities. One of the runoffs was more extreme with regards to pH and higher concentration of soluble iron and heavy metals. These conditions favored the development of algal-dominated microbial mats. Typical AMD microorganisms related to known iron-oxidizing bacteria (Acidithiobacillus ferrivorans, Acidobacteria and Actinobacteria) dominated the bacterial community although some unexpected populations related to Chloroflexi were also significant. No microbial mats were found in the second area. The geochemistry here showed less extreme drainage, most likely in direct contact with the ore under the waste pile. Large deposits of secondary minerals were found and the presence of iron stalks was revealed by microscopy analysis. Although typical AMD microorganisms were also detected here, the microbial community was dominated by other populations, some of them new to this type of system (Saccharibacteria, Gallionellaceae). These were absent or lowered in numbers the farther from the spring source and they could represent native populations involved in the oxidation of sulphide rocks within the waste rock pile. This environment appears thus as a highly interesting field of potential novelty in terms of both phylogenetic/taxonomic and functional diversity.

Comparative microbial ecology of the water column of an extreme acidic pit lake, Nuestra Señora del Carmen, and the Río Tinto basin (Iberian Pyrite Belt)

Summary. The Iberian Pyrite Belt, located in Southwestern Spain, represents one of the world’s largest accumulations of mine wastes and acid mine drainages. This study reports the comparative microbial ecology of the water column of Nuestra Senora del Carmen acid pit lake with the extreme acidic Rio Tinto basin. The canonical correspondence analysis identified members of the Leptospirillum, Acidiphilium, Metallibacterium, Acidithiobacillus, Ferrimicrobium and Acidisphaera genera as the most representative microorganisms of both ecosystems. The presence of archaeal members is scarce in both systems. Only sequences clustering with the Thermoplasmata have been retrieved in the bottom layer of Nuestra Senora del Carmen and one station of Rio Tinto. Although the photosynthetically active radiation values measured in this lake upper layer were low, they were sufficient to activate photosynthesis in acidophilic microorganisms. All identified photosynthetic microorganisms in Nuestra Senora del Carmen (members of the Chlamydomonas, Zygnemopsis and Klebsormidium genera) are major members of the photosynthetic eukaryotic community characterized in Rio Tinto basin. This study demonstrates a close relationship between the microbial diversity of Nuestra Senora del Carmen pit lake and the diversity detected in the Rio Tinto basin, which underlain the influence of the shared mineral substrates in the microbial ecology of these ecosystems. [Int Microbiol 2014; 17(4):225233]

Characterization of the Anoxic Sediments of Rio Tinto: Biohydrometallurgical Implications

Rio Tinto, a natural extreme acidic environment with a rather constant acidic pH and a high concentration of heavy metals, is the product of the metabolic activity of chemolithotrophic microorganisms thriving in the rich complex sulfides of the Iberian Pyritic Belt. Up to now the microbial characterization has been made mainly in the water column and biofilm samples. Since all ferric reducing activities will not benefit the bioleaching operations, we consider it critical to ascertain the ecology and metabolic properties of the microorganisms inhabiting the anoxic part of the sediments, to facilitate the design and control the operation of heap bioleaching processes, maximizing their efficiency. The implication of these microorganisms in biohydrometallurgical operations is discussed.

Microbial Ecology of a Natural Extreme Acidic Environment: Lessons from Río Tinto

Rio Tinto (Iberian Pyritic Belt, SW Spain) is a natural extreme acidic environment with a rather constant acidic pH (mean pH value 2.3) and a high concentration of heavy metals. The Tinto ecosystem is under the control of iron. The geomicrobiological characterization of Río Tinto has unravelled some basic questions of biohydrometallurgical interest. The methodologies developed for this study were applied successfully to monitor different bioleaching processes of the BioMinE project.

Diversity Patterns and Isolation of Planctomycetes Associated with Metalliferous Deposits from Hydrothermal Vent Fields Along the Valu Fa Ridge (SW Pacific)

The microbial diversity associated with diffuse venting deep-sea hydrothermal deposits is tightly coupled to the geochemistry of the hydrothermal fluids. Previous 16S rRNA gene amplicon sequencing (metabarcoding) of marine iron-hydroxide deposits along the Arctic Mid Ocean Ridge, revealed the presence of diverse bacterial communities associated with these deposits (Storesund and Øvreås in Antonie van Leeuwenhoek 104:569–584, 2013). One of the most abundant and diverse phyla detected was the enigmatic Planctomycetes. Here we report on the comparative analyses of the diversity and distribution patterns of Planctomycetes associated with metalliferous deposits from two diffuse-flow hydrothermal vent fields (Mariner and Vai Lili) from the Valu Fa Ridge in the Southwestern Pacific. Metabarcoding of 16S rRNA genes showed that the major prokaryotic phyla were Proteobacteria (51–73% of all 16S rRNA gene reads), Epsilonbacteraeota (0.5–19%), Bacteriodetes (5–17%), Planctomycetes (0.4–11%), Candidatus Latescibacteria (0–5%) and Marine Benthic Group E (Hydrothermarchaeota) (0–5%). The two different sampling sites differed considerably in overall community composition. The abundance of Planctomycetes also varied substantially between the samples and the sites, with the majority of the sequences affiliated with uncultivated members of the classes Planctomycetacia and Phycisphaerae, and other deep branching lineages. Seven different strains affiliated with the order Planctomycetales were isolated, mostly from the Vai Lili samples, where also the highest Planctomycetales diversity was seen. Most of the isolates were affiliated with the genera Gimesia, Rhodopirellula and Blastopirellula. One isolate was only distantly related to known cultured, but uncharacterized species within the Pir4 group. This study shows that the deep-sea Planctomycetes represent a very heterogeneous group with a high phylogenetic diversity and a substantial potential for novel organism discovery in these deep ocean environments.

Microbial ecology of Leptospirillum spp. in Río Tinto, a model of interest to biohydrometallurgy

Members of Leptospirillum genus have emerged not only as one of the most representative bacteria in the Río Tinto ecosystem, but also in other acidic environments (AMD), and in biohydrometallurgical operations. The main objective of this work was to study the role of chemolithoautotrophic bacteria of the genus Leptospirillum in the Río Tinto iron cycle (an extreme acidic environment, characterized by its constant low pH) to better understand and control industrial biohydrometallurgical processes. Different strains of Leptospirillum were isolated from the Río Tinto basin and physiologically and genetically characterized. Certain metabolic capabilities, such as pyrite leaching, iron oxidation and nitrogen fixation, were determined for each strain. Complementary molecular ecology techniques (FISH, CARD-FISH and cloning) were used to study the microbial diversity and the distribution of leptospirilli along the iron gradient in the different phases of Río Tinto: water column, anaerobic sediments and biofilms.