Ancient Microbes from Halite Fluid Inclusions: Optimized Surface Sterilization and DNA Extraction

Fluid inclusions in evaporite minerals (halite, gypsum, etc.) potentially preserve genetic records of microbial diversity and changing environmental conditions of Earth's hydrosphere for nearly one billion years. Here we describe a robust protocol for surface sterilization and retrieval of DNA from fluid inclusions in halite that, unlike previously published methods, guarantees removal of potentially contaminating surface-bound DNA. The protocol involves microscopic visualization of cell structures, deliberate surface contamination followed by surface sterilization with acid and bleach washes, and DNA extraction using Amicon centrifugal filters. Methods were verified on halite crystals of four different ages from Saline Valley, California (modern, 36 ka, 64 ka, and 150 ka), with retrieval of algal and archaeal DNA, and characterization of the algal community using ITS1 sequences. The protocol we developed opens up new avenues for study of ancient microbial ecosystems in fluid inclusions, understanding microbial evolution across geological time, and investigating the antiquity of life on earth and other parts of the solar system

High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study.

12 páginas, 2 tablas, 5 figures.We surveyed the archaeal assemblage in a stratified sulfurous lake (Lake Vilar, Banyoles, Spain) over 5 consecutive years to detect potential seasonal and interannual trends in the free-living planktonic Archaea composition. The combination of different primer pairs and nested PCR steps revealed an unexpectedly rich archaeal community. Overall, 140 samples were analyzed, yielding 169 different 16S rRNA gene sequences spread over 14 Crenarchaeota (109 sequences) and six Euryarchaeota phylogenetic clusters. Most of the Crenarchaeota (98% of the total crenarchaeotal sequences) affiliated within the Miscellaneous Crenarchaeota Group (MCG) and were related to both marine and freshwater phylotypes. Euryarchaeota mainly grouped within the Deep Hydrothermal Vent Euryarchaeota (DHVE) cluster (80% of the euryarchaeotal sequences) and the remaining 20% distributed into three less abundant taxa, most of them composed of soil and sediment clones. The largest fraction of phylotypes from the two archaeal kingdoms (79% of the Crenarchaeota and 54% of the Euryarchaeota) was retrieved from the anoxic hypolimnion, indicating that these cold and sulfide-rich waters constitute an unexplored source of archaeal richness. The taxon rank-frequency distribution showed two abundant taxa (MCG and DHVE) that persisted in the water column through seasons, plus several rare ones that were only detected occasionally. Differences in richness distribution and seasonality were observed, but no clear correlations were obtained when multivariate statistical analyses were carried out.This study was funded through projects VIARC (Ref. REN 2003-08333-GLO) and CRENYC (Ref. CGL2006-12058-BOS) to C.M.B. and E.O.C. from the Spanish Ministerio de Educación y Ciencia (MEC). M.L. is recipient of a PhD student fellowship (BES-2004-5127) from the Spanish MEC.Peer reviewe

Maintenance of previously uncultured freshwater archaea from anoxic waters under laboratory conditions

6 páginas, 2 figuras, 2 tablas.Culture conditions for the maintenance of previously uncultured members of the Archaea thriving in anoxic water layers of stratified freshwater lakes are described. The proposed enrichment conditions, based on the use of defined medium composition and the maintenance of anoxia, have been proven effective for the maintenance of the archaeal community with virtually no changes over time for periods up to 6 months as revealed by a PCR-DGGE analysis. Phylotypes belonging to groups poorly represented in culture collections such as the DeepSea Hydrothermal Vent Euryarchaeota (DHVE) and the Miscellaneous Crenarchaeotic Group (MCG) were maintained and selectively enriched when compared to the correspondent indigenous planktonic archaeal community.This work was supported by coordinated projects REN2003-08333 and CRENYC CGL2006-12058 from the Spanish Ministerio de Educación y Ciencia (MEC) to CMB and EOC.Peer reviewe

Contribution of deep dark fixation processes to overall CO2 incorporation and large vertical changes of microbial populations in stratified karstic lakes.

15 páginas, 7 figuras, 2 tablas.We carried out a detailed study in five stratified lakes in the karstic regions of NE Spain along a redox gradient combining vertical profiles of inorganic carbon dioxide fixation and analysis of microbial (bacteria and archaea) community composition determined by 16S rRNA gene fingerprinting (DGGE), microscopic counts, and pigment analysis. High rates of non-photosynthetic (i.e., ‘‘dark’’) inorganic carbon incorporation were detected mostly at deeper layers after short-term in situ incubations at noon. Significant contribution of dark CO2 incorporation was observed at the whole lake level for the single time sampling, ranging between 4 and 19% of total carbon fixation measured, and up to 31% in the case of a meromictic basin. Good agreement was found between vertical patterns in redox conditions and the different microbial diversity descriptors (DGGE band sequencing, microscopic analysis, and pigment data), showing large vertical changes in microbial community composition covering a wide range of phylogenetic diversity. Cyanobacteria, Alpha and Beta-Proteobacteria, Actinobacteria, Flavobacteria and Flectobacillaceae were the most frequently recovered groups in the DGGE from oxygenated water masses. In anoxic waters, we found Beta-Proteobacteria mostly of the Rhodoferax group, Gamma-Proteobacteria (Chromatiaceae), Delta-Proteobacteria related to different sulfate reducing bacteria, Chlorobiaceae, and anaerobic Bacteroidetes spread among the Bacteroidales, Flavobacteriales and Saprospiraceae. However, as a whole, we did not find any significant correlation between dark fixation rates and either nutrient distribution and microbial community composition in the study lakes. All of this suggests that (1) different physiologies and ecologies are simultaneously contributing to the process (2) more sensitive methods are needed and more specific compounds measured and (3) some of the non-specialist microbial populations detected may carry out carbon dioxide assimilation in the dark under in situ conditions.This research was supported by projects VIARC REN2003-08333 and CRENYC CGL2006-12058 to EOC and CBM, from the Spanish Ministerio de Ciencia e Innovacio´n (MICINN), and CONSOLIDER-INGENIO 2010 project GRACCIE CSD2007-00067 from MICINN.Peer reviewe

Pelagic photoferrotrophy and iron cycling in a modern ferruginous basin

8 páginas, 3 figurasIron-rich (ferruginous) ocean chemistry prevailed throughout most of Earth’s early history. Before the evolution and proliferation of oxygenic photosynthesis, biological production in the ferruginous oceans was likely driven by photoferrotrophic bacteria that oxidize ferrous iron {Fe(II)} to harness energy from sunlight, and fix inorganic carbon into biomass. Photoferrotrophs may thus have fuelled Earth’s early biosphere providing energy to drive microbial growth and evolution over billions of years. Yet, photoferrotrophic activity has remained largely elusive on the modern Earth, leaving models for early biological production untested and imperative ecological context for the evolution of life missing. Here, we show that an active community of pelagic photoferrotrophs comprises up to 30% of the total microbial community in illuminated ferruginous waters of Kabuno Bay (KB), East Africa (DR Congo). These photoferrotrophs produce oxidized iron {Fe(III)} and biomass, and support a diverse pelagic microbial community including heterotrophic Fe(III)-reducers, sulfate reducers, fermenters and methanogens. At modest light levels, rates of photoferrotrophy in KB exceed those predicted for early Earth primary production, and are sufficient to generate Earth’s largest sedimentary iron ore deposits. Fe cycling, however, is efficient, and complex microbial community interactions likely regulate Fe(III) and organic matter export from the photic zone.This work was partially supported by Belgian (FNRS 2.4.515.11 and BELSPO SD/AR/02A contracts), Danish (grant no. DNRF53 to DEC), and European (grant no. ERC-StG 240002, for stable isotope measurements) funds. AVB is a senior research associate at the FRS-FNRS. SAC was supported by the Agouron institute.Peer reviewe