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Bones are an underexploited, yet potentially profitable feedstock for biotechnological advances and value chains, due to the sheer amounts of residues produced by the modern meat and poultry processing industry. In this metagenomic study, we decipher the microbial pathways and enzymes that we postulate to be involved in bone degradation in the marine environment., The marine bone biome is a complex assemblage of macro- and microorganisms; however, the enzymatic repertoire to access bone-derived nutrients remains unknown. The bone matrix is a composite material made up mainly of organic collagen and inorganic hydroxyapatite. We conducted field experiments to study microbial assemblages that can use organic bone components as nutrient source. Bovine and turkey bones were deposited at 69 m depth in a Norwegian fjord (Byfjorden, Bergen). Metagenomic sequence analysis was used to assess the functional potential of microbial assemblages from bone surface and the bone-eating worm Osedax mucofloris, which is a frequent colonizer of whale falls and known to degrade bone. The bone microbiome displayed a surprising taxonomic diversity revealed by the examination of 59 high-quality metagenome-assembled genomes from at least 23 bacterial families. Over 700 genes encoding enzymes from 12 relevant enzymatic families pertaining to collagenases, peptidases, and glycosidases putatively involved in bone degradation were identified. Metagenome-assembled genomes (MAGs) of the class Bacteroidia contained the most diverse gene repertoires. We postulate that demineralization of inorganic bone components is achieved by a timely succession of a closed sulfur biogeochemical cycle between sulfur-oxidizing and sulfur-reducing bacteria, causing a drop in pH and subsequent enzymatic processing of organic components in the bone surface communities. An unusually large and novel collagen utilization gene cluster was retrieved from one genome belonging to the gammaproteobacterial genus Colwellia. IMPORTANCE Bones are an underexploited, yet potentially profitable feedstock for biotechnological advances and value chains, due to the sheer amounts of residues produced by the modern meat and poultry processing industry. In this metagenomic study, we decipher the microbial pathways and enzymes that we postulate to be involved in bone degradation in the marine environment. We here demonstrate the interplay between different bacterial community members, each supplying different enzymatic functions with the potential to cover an array of reactions relating to the degradation of bone matrix components. We identify and describe a novel gene cluster for collagen utilization, which is a key function in this unique environment. We propose that the interplay between the different microbial taxa is necessary to achieve the complex task of bone degradation in the marine environment.

[EN] To support the bio-based industry in development of environment-friendly processes and products, an optimal toolbox of biocatalysts is key. Although functional screen of (meta)genomic libraries may potentially contribute to identifying new enzymes, the discovery of new enzymes meeting industry compliance demands is still challenging. This is particularly noticeable in the case of proteases, for which the reports of metagenome-derived proteases with industrial applicability are surprisingly limited. Indeed, proteolytic clones have been typically assessed by its sole activity on casein or skim milk and limited to mild screening conditions. Here, we demonstrate the use of six industry-relevant animal and plant by-products, namely bone, feather, blood meals, gelatin, gluten, and zein, as complementary substrates in functional screens and show the utility of temperature as a screening parameter to potentially discover new broad-substrate range and robust proteases for the biorefinery industry. By targeting 340,000 clones from two libraries of pooled isolates of mesophilic and thermophilic marine bacteria and two libraries of microbial communities inhabiting marine environments, we identified proteases in four of eleven selected clones that showed activity against all substrates herein tested after prolonged incubation at 55 °C. Following sequencing, in silico analysis and recombinant expression in Escherichia coli, one functional protease, 58% identical at sequence level to previously reported homologs, was found to readily hydrolyze highly insoluble zein at temperatures up to 50 °C and pH 9–11. It is derived from a bacterial group whose ability to degrade zein was unknown. This study reports a two-step screen resulting in identification of a new marine metagenome-derived protease with zein-hydrolytic properties at common biomass processing temperatures that could be useful for the modern biorefinery industry. Key points: • A two-step multi-substrate strategy for discovery of robust proteases. • Feasible approach for shortening enzyme optimization to industrial demands. • A new temperature-tolerant protease efficiently hydrolyzes insoluble zein., Open access funding provided by NORCE Norwegian Research Centre AS. This work was funded by “INMARE” under the European Union’s Horizon 2020 programme (grant agreement no. 634486) and the Marine Biotechnology ERA-NET and from the Ministerio de Economía y Competitividad, Ministerio de Ciencia,Innovación y Universidades, Agencia Estatal de Investigación, Fondo Europeo de Desarrollo Regional (grants PCIN-2017-078 and BIO2017-85522-R).

We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus’ capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host., Since its discovery as part of the bacterial adaptative immune system, CRISPR/Cas has emerged as the most promising tool for targeted genome editing over the past few years. Various tools for genome editing in Bacillus subtilis have recently been developed, expanding and simplifying its potential development as an industrial species. A collection of vectors compatible with high-throughput (HTP) fragment exchange (FX) cloning for heterologous expression in Escherichia coli and Bacillus was previously developed. This vector catalogue was through this work supplemented with editing plasmids for genome engineering in Bacillus by adapting two CRISPR/Cas plasmids to the cloning technology. The customized tools allow versatile editing at any chosen genomic position (single-plasmid strategy) or at a fixed genomic locus (double-plasmid strategy). The single-plasmid strategy was validated by deleting the spoIIAC gene, which has an essential role in sporulation. Using the double-plasmid strategy, we demonstrate the quick transition from plasmid-based subtilisin expression to the stable integration of the gene into the amyE locus of a seven-protease-deficient KO7 strain. The newly engineered B. subtilis strain allowed the successful production of a functional enzyme. The customized tools provide improvements to the cloning procedure, should be useful for versatile genomic engineering, and contribute to a cloning platform for a quick transition from HTP enzyme expression to production through the fermentation of industrially relevant B. subtilis and related strains. IMPORTANCE We complemented a cloning platform with new editing plasmids that allow a quick transition from high-throughput cloning and the expression of new enzymes to the stable integration of genes for the production of enzymes through B. subtilis fermentation. We present two systems for the effective assembly cloning of any genome-editing cassette that shortens the engineering procedure to obtain the final editing constructs. The utility of the customized tools is demonstrated by disrupting Bacillus’ capacity to sporulate and by introducing the stable expression of subtilisin. The tools should be useful to engineer B. subtilis strains by a variety of recombination events to ultimately improve the application range of this industry-relevant host.

Enzyme-based conversion of marine biomass to high-quality peptide ingredients leaves a distinct smell of “fish” caused by the presence of trimethylamine, which limits their economic potential. We suggest an enzymatic solution for converting trimethylamine to the odorless trimethylamine N-oxide as a novel strategy to improve the smell quality of marine protein hydrolysates. Following a systematic investigation of 45 putative bacterial trimethylamine monooxygenases from several phyla, we expand the repertoire of known active trimethylamine monooxygenases. As a proof-of-concept, we demonstrate that three of these enzymes oxidized trimethylamine in an industry-relevant salmon protein hydrolysate. Our results add new oxidoreductases to the industrial biocatalytic toolbox and provide a new point of departure for enzyme process developments in marine biorefineries., Enzymatic processing of fish by-products for recovery of peptides (hydrolysates) is a promising technology to reach food grade ingredients of high nutritional quality. Despite this, their bitter taste and “fish” odor block implementation in food products and limit their economic potential. Trimethylamine (TMA) is a known contributor to malodor in fish. Current strategies to mask or remove the odor either are not effective or give rise to undesirable side effects. As an alternative approach to remediate TMA, we propose a novel enzymatic strategy to convert TMA into the odorless trimethylamine N-oxide (TMAO) using TMA monooxygenases (Tmms). We identified a diverse set of bacterial Tmms using a sequence similarity network. Purified, recombinant enzymes were assessed for their biocatalytic capacity by monitoring NADPH consumption and TMAO generation. Selected Tmms were subjected to biochemical characterization and investigated for their ability to oxidize TMA in an industry-relevant substrate. From the 45 bacterial Tmm candidates investigated, eight enzymes from four different taxa were selected for their high activity toward TMA. The three most active enzymes were shown to vary in temperature optimum, with the highest being 45°C. Enzymatic activity dropped at high temperatures, likely due to structural unfolding. The enzymes were all active from pH 6.0 to 8.5, with functional stability being lowest around the optimal pH. All three Tmms, given sufficient NADPH cofactor, were found to generate TMAO in the TMA-rich salmon protein hydrolysate. The Tmms serve as unique starting points for engineering and should be useful for guiding process development for marine biorefineries. IMPORTANCE Enzyme-based conversion of marine biomass to high-quality peptide ingredients leaves a distinct smell of “fish” caused by the presence of trimethylamine, which limits their economic potential. We suggest an enzymatic solution for converting trimethylamine to the odorless trimethylamine N-oxide as a novel strategy to improve the smell quality of marine protein hydrolysates. Following a systematic investigation of 45 putative bacterial trimethylamine monooxygenases from several phyla, we expand the repertoire of known active trimethylamine monooxygenases. As a proof-of-concept, we demonstrate that three of these enzymes oxidized trimethylamine in an industry-relevant salmon protein hydrolysate. Our results add new oxidoreductases to the industrial biocatalytic toolbox and provide a new point of departure for enzyme process developments in marine biorefineries.

The marine bone biome is a complex assemblage of macro- and microorganisms, however the enzymatic repertoire to access bone-derived nutrients remains unknown. The bone matrix is a composite material made up mainly of organic collagen and inorganic hydroxyapatite. We conducted field experiments to study microbial assemblages that can use organic bone components as nutrient source. Bovine and turkey bones were deposited at 69 m depth in a Norwegian fjord (Byfjorden, Bergen). Metagenomic sequence analysis was used to assess the functional potential of microbial assemblages from bone surface and the bone eating worm Osedax mucofloris, which is a frequent colonizer of whale falls and known to degrade bone. The bone microbiome displayed a surprising taxonomic diversity revealed by the examination of 59 high-quality metagenome assembled genomes from at least 23 bacterial families. Over 700 genes encoding enzymes from twelve relevant enzymatic families pertaining to collagenases, peptidases, glycosidases putatively involved in bone degradation were identified. Metagenome assembled genomes (MAGs) of the class Bacteroidia contained the most diverse gene repertoires. We postulate that demineralization of inorganic bone components is achieved by a timely succession of a closed sulfur biogeochemical cycle between sulfur-oxidizing and sulfur-reducing bacteria, causing a drop in pH and subsequent enzymatic processing of organic components in the bone surface communities. An unusually large and novel collagen utilization gene cluster was retrieved from one genome belonging to the gammaproteobacterial genus Colwellia.ImportanceBones are an underexploited, yet potentially profitable feedstock for biotechnological advances and value chains, due to the sheer amounts of residues produced by the modern meat and poultry processing industry. In this metagenomic study we decipher the microbial pathways and enzymes that we postulate to be involved in bone degradation marine environment. We herein demonstrate the interplay between different bacterial community members, each supplying different enzymatic functions with the potential to cover an array of reactions relating to the degradation of bone matrix components. We identify and describe a novel gene cluster for collagen utilization, which is a key function in this unique environment. We propose that the interplay between the different microbial taxa is necessary to achieve the complex task of bone degradation in the marine environment.

Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E. coli, and is active in presence of calcium, which is thought to have a role in minor, but essential, structural rearrangements needed for catalytic activity. The ISP operates at alkaline pH and at moderate temperatures, and has a corresponding melting temperature around 60 °C. The high-resolution 3-dimensional structure reported here, represents an ISP with an intact catalytic triad albeit in a configuration with an inhibitory pro-peptide bound. The pro-peptide is removed in other homologs, but the removal of the pro-peptide from the Planococcus sp. AW02J18 ISP appears to be different, and possibly involves several steps. A first processing step is described here as the removal of 2 immediate N-terminal residues. Furthermore, the pro-peptide contains a conserved LIPY/F-motif, which was found to be involved in inhibition of the catalytic activity.

Aerobic moderately thermophilic and thermophilic methane-oxidizing bacteria make a substantial contribution in the control of global warming through biological reduction of methane emissions and have a unique capability of utilizing methane as their sole carbon and energy source. Here, we report a novel moderately thermophilic Methylococcus-like Type Ib methanotroph recovered from an alkaline thermal spring (55.4 &deg, C and pH 8.82) in the Ethiopian Rift Valley. The isolate, designated LS7-MC, most probably represents a novel species of a new genus in the family Methylococcaceae of the class Gammaproteobacteria. The 16S rRNA gene phylogeny indicated that strain LS7-MC is distantly related to the closest described relative, Methylococcus capsulatus (92.7% sequence identity). Growth was observed at temperatures of 30&ndash, 60 &deg, C (optimal, 51&ndash, 55 &deg, C), and the cells possessed Type I intracellular membrane (ICM). The comparison of the pmoA gene sequences showed that the strain was most closely related to M. capsulatus (87.8%). Soluble methane monooxygenase (sMMO) was not detected, signifying the biological oxidation process from methane to methanol by the particulate methane monooxygenase (pMMO). The other functional genes mxaF, cbbL and nifH were detected by PCR. To our knowledge, the new strain is the first isolated moderately thermophilic methanotroph from an alkaline thermal spring of the family Methylococcaceae. Furthermore, LS7-MC represents a previously unrecognized biological methane sink in thermal habitats, expanding our knowledge of its ecological role in methane cycling and aerobic methanotrophy.

Eukarya have been discovered in the deep subsurface at several locations in South Africa, but how organisms reach the subsurface remains unknown. We studied river-subsurface fissure water systems and identified Eukarya from a river that are genetically identical for 18S rDNA. To further confirm that these are identical species one metazoan species recovered from the overlying river interbred successfully with specimen recovered from an underlying mine at −1.4 km. In situ seismic simulation experiments were carried out and show seismic activity to be a major force increasing the hydraulic conductivity in faults allowing organisms to create ecosystems in the deep subsurface. As seismic activity is a non-selective force we recovered specimen of algae and Insecta that defy any obvious other explanation at a depth of −3.4 km. Our results show there is a steady flow of surface organisms to the deep subsurface where some survive and adapt and others perish. As seismic activity is also present on other planets and moons in our solar system the mechanism elucidated here may be relevant for future search and selection of landing sites in planetary exploration.

A global census of marine microbial life has been underway over the past several decades. During this period, there have been scientific breakthroughs in estimating microbial diversity and understanding microbial functioning and ecology. It is estimated that the ocean, covering 71% of the earth's surface with its estimated volume of about 2 × 1018 m3 and an average depth of 3800 m, hosts the largest population of microbes on Earth. More than 2 million eukaryotic and prokaryotic species are thought to thrive both in the ocean and on its surface. Prokaryotic cell abundances can reach densities of up to 1012 cells per millilitre, exceeding eukaryotic densities of around 106 cells per millilitre of seawater. Besides their large numbers and abundance, marine microbial assemblages and their organic catalysts (enzymes) have a largely underestimated value for their use in the development of industrial products and processes. In this perspective article, we identified critical gaps in knowledge and technology to fast-track this development. We provided a general overview of the presumptive microbial assemblages in oceans, and an estimation of what is known and the enzymes that have been currently retrieved. We also discussed recent advances made in this area by the collaborative European Horizon 2020 project ‘INMARE’., The success of using marine resources for the development of industrial products and processes depends on the manner they are screened, produced and tested on a larger scale.

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., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Intracellular subtilisin proteases (ISPs) have important roles in protein processing during the stationary phase in bacteria. Their unregulated protein degrading activity may have adverse effects inside a cell, but little is known about their regulatory mechanism. Until now, ISPs have mostly been described from Bacillus species, with structural data from a single homolog. Here, we study a marine ISP originating from a phylogenetically distinct genus, Planococcus sp. The enzyme was successfully overexpressed in E. coli, and is active in presence of calcium, which is thought to have a role in minor, but essential, structural rearrangements needed for catalytic activity. The ISP operates at alkaline pH and at moderate temperatures, and has a corresponding melting temperature around 60 °C. The high‐resolution 3‐dimensional structure reported here, represents an ISP with an intact catalytic triad albeit in a configuration with an inhibitory pro‐peptide bound. The pro‐peptide is removed in other homologs, but the removal of the pro‐peptide from the Planococcus sp. AW02J18 ISP appears to be different, and possibly involves several steps. A first processing step is described here as the removal of 2 immediate N‐terminal residues. Furthermore, the pro‐peptide contains a conserved LIPY/F‐motif, which was found to be involved in inhibition of the catalytic activity.

Substrate specificity and selectivity of a biocatalyst are determined by the protein sequence and structure of its active site. Finding versatile biocatalysts acting against multiple substrates while at the same time being chiral selective is of interest for the pharmaceutical and chemical industry. However, the relationships between these two properties in natural microbial enzymes remain underexplored. Here, we performed an experimental analysis of substrate promiscuity and chiral selectivity in a set of 145 purified esterases from phylogenetically and environmentally diverse microorganisms, which were assayed against 96 diverse esters, 20 of which were enantiomers. Our results revealed a negative correlation between substrate promiscuity and chiral selectivity in the evaluated enzymes. Esterases displaying prominent substrate promiscuity and large catalytic environments are characterized by low chiral selectivity, a feature that has limited commercial value. Although a low level of substrate promiscuity does not guarantee high chiral selectivity, the probability that esterases with smaller active sites possess chiral selectivity factors of interest for industry (>25) is significantly higher than for promiscuous enzymes. Together, the present study unambiguously demonstrates that promiscuous and selective esterases appear to be rare in nature and that substrate promiscuity can be used as an indicator of the chiral selectivity level of esterases, and vice versa.

Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases’ substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein–ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets., C.C. thanks the Spanish Ministry of Economy, Industry and Competitiveness for a Ph.D. fellowship (Grant BES-2015-073829). This project received funding from the European Union’s Horizon 2020 research and innovation program [Blue Growth: Unlocking the potential of Seas and Oceans] under grant agreement no. 634486 (project acronym INMARE). This research was also supported by the European Community Projects MAGICPAH (FP7-KBBE-2009-245226), ULIXES (FP7-KBBE-2010-266473), and KILLSPILL (FP7-KBBE2012-312139) and grants BIO2011-25012, PCIN-2014-107, BIO2014-54494-R, and CTQ2016-79138-R from the Spanish Ministry of Economy, Industry and Competitiveness. The present investigation was also funded by the Spanish Ministry of Economy, Industry and Competitiveness within the ERA NET IB2, grant no. ERA-IB-14-030 (MetaCat), the UK Biotechnology and Biological Sciences Research Council (BBSRC), grant no. BB/M029085/1, and the German Research Foundation (FOR1296). R.B. and P.N.G. acknowledge the support of the Supercomputing Wales project, which is part-funded by the European Regional Development Fund (ERDF) via the Welsh Government. O.V.G. and P.N.G. acknowledge the support of the Centre of Environmental Biotechnology Project funded by the European Regional Development Fund (ERDF) through the Welsh Government. A.Y. and A.S. gratefully acknowledge funding from Genome Canada (2009-OGI-ABC-1405) and the NSERC Strategic Network grant IBN. A.I.P. was supported by the Counseling of Economy and Employment of the Principality of Asturias, Spain (Grant FC-15-GRUPIN14-107). V.G. acknowledges the joint BSC-CRG-IRB Research Program in Computational Biology. The authors gratefully acknowledge financial support provided by the European Regional Development Fund (ERDF)., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

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 Ovreas 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.

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., Spanish Ministry of Science and Innovation (CGL2008-02298/BOS, CGL2011-22540)

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(1-4). 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.

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

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.

ARTICLE I NFO Members of the Leptospirillum genus have recently been found to be not only the most representative prokaryotes in the Rio Tinto, a natural extreme acidic habitat, but also in other acidic environments (AMD), as well as in biohydrometallurgical operations. The main objective of this work was to better understand and control industrial biohydrometallurgical processes by studying the role of chemolithoautotrophic bacteria of this genus in the Tinto ecosystem. Different strains of Leptospirillum were isolated from the Tinto basin and physiologically and genetically characterized by Pulsed Field Gel Electrophoresis (PFGE) and DNA microarrays. Certain metabolic capabilities, such as iron oxidation, pyrite leaching and nitrogen fixation, were determined for each strain. Complementary molecular ecology techniques (fluorescence in situ hybridization (FISH and CARD-FISH) and 16S rRNA gene cloning) were used to study the microbial diversity and the distribution of leptospirilli along the iron gradient of the Tinto ecosystem.

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.

12 pages, 5 figures, 2 tables.-- ISI Article Identifier: 000244695500010.-- Available online Sep 28, 2006.-- Issue title: "Planet Mars II"., The geomicrobiological characterization of the Río Tinto (Iberian Pyritic Belt) has recently proven the importance of the iron cycle, not only in the generation of the extreme conditions of the habitat (low pH, high concentration of heavy metals), but also in the maintenance of a high level of microbial diversity. The presence of vast deposits of sulfates and iron oxides on Mars, the main products of the bioleaching of iron containing sulfides found in Río Tinto, and the physico-chemical properties of iron as a source of energy, protection from radiation and oxidative stress as well as pH control, make Río Tinto an interesting Mars terrestrial analog., This work was supported by Grant BOS2002-02148 from the CICYT and Institutional Grants to the Centro de Astrobiología.

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 Señora del Carmen acid pit lake with the extreme acidic Río 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 Señora del Carmen and one station of Río 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 Señora del Carmen (members of the Chlamydomonas, Zygnemopsis and Klebsormidium genera) are major members of the photosynthetic eukaryotic community characterized in Río Tinto basin. This study demonstrates a close relationship between the microbial diversity of Nuestra Señora del Carmen pit lake and the diversity detected in the Río Tinto basin, which underlain the influence of the shared mineral substrates in the microbial ecology of these ecosystems.

The recent geomicrobiological characterization of Rio Tinto, Iberian Pyrite Belt (IPB), 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, both prokaryotic and eukaryotic, detected in the water column and the sediments. The extreme conditions of 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 IPB. To test this hypothesis, a drilling project was carried out to intersect ground waters that interact with the mineral ore in order to provide evidence of subsurface microbial activities and the potential resources to support these activities. The oxidants that drive the system appear to come from the rock matrix, contradicting conventional acid mine drainage models. These resources need only groundwater to launch microbial metabolism. There are several similarities between the vast deposits of sulfates and iron oxides on Mars and the main sulfide-containing iron bioleaching products found in the Tinto. Firstly, the short-lived methane detected both in Mars' atmosphere and in the sediments and subsurface of the IPB and secondly, the abundance of iron, common to both. The physicochemical properties of iron make it a source of energy, a shield against radiation and oxidative stress as well as a natural pH controller. These similarities have led to Rio Tinto's status as a Mars terrestrial analogue.

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.

14 pages, 6 figures, 3 tables.-- PMID: 17950555 [PubMed].-- Printed version published Dec 6, 2007.-- ISI Article Identifier: 000251655600003., The prokaryotic diversity of macroscopic filaments located at the water surface in an extreme acidic environment, Río Tinto (SW, Spain), has been analysed through denaturing gradient gel electrophoresis (DGGE), cloning of 16S rRNA genes and fluorescence in situ hybridization (FISH). The dominant species present in the macrofilaments were Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans and Acidiphilium spp., which represented the three main bacterial genera found in the water column of the river. However, our results also showed the presence of other microorganisms not previously detected in the Río Tinto. Within the Gammaproteobacteria class, sequences closely related to the iron-oxidizing bacteria WJ2 and DSM 2392 were found, as well as sequences related to the non-acidophilic genera Aeromonas and Acinetobacter. In addition, two other new phylotypes related to Gram-positive species from the genera Desulfosporosinus, Clostridium and Mycobacterium were identified. The presence of these anaerobic microorganisms strongly suggests that the filaments could originate in the deeper parts of the river as a typical acid streamer attached to the rocks or the sediments and be pulled up towards the surface when they reach certain buoyancy., This research was supported by the Instituto de Técnica Aeroespacial (INTA), a Grant from the Ministerio de Educación y Ciencia (MEC) (CGL2006-02534/BOS) and an institutional Grant from Fundación Areces to the CBMSO. GM is a MEC predoctoral fellow, EGT is an INTA postdoctoral fellow and AA a Ramón y Cajal fellow from the MEC. The authors also thank Juan Pérez Mercader for his continuous support.