Chloroflexus-like organisms from marine and hypersaline environments: Distribution and diversity

We report the presence of a diverse number ofChloroflexus-like organisms in intertidal marine and submerged hypersaline microbial mats using light, infrared fluorescence, and electron microscopy. The intertidal organisms appear morphologically very similar to thermophilicC. aurantiacus while the 2 hypersaline strains are larger and have a more complex ultrastructure composed of chlorosome-bearing internal membranes that appear to arise as invaginations of the cell membrane. By comparing spectroradiometry of microbial mat layers with microscopic observations, we have confirmed that theChloroflexus-like organisms are major constituents of the hypersaline microbial mat communities. In situ studies on mat layers dominated byChloroflexus-like organisms showed that sulfide-dependent photoautotrophic activity sustained by near infrared radiation prevailed. Autoradiographic analyses revealed that autotrophy was sustained in the filaments by 750 nm radiation. Three morphologically distinct strains are now maintained in mixed culture. One of these appears to be growing photoautotrophically.     

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.     

Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

Structural and Geomicrobiological Characteristics of a Microbial Community from a Cold Sulfide Spring

The Ancaster sulfur spring is a cold (9°C) sulfur spring located near Ancaster, Ontario, Canada, which hosts an abundant and diverse microbial mat community. We conducted an extensive microscopical study of the microbial community of this spring using a number of techniques: phase light, confocal scanning laser microscopy, conventional scanning electron microscopy using both chemical/critical point drying and cryofixation preparative techniques, environmental scanning electron microscopy, and transmission electron microscopy. The latter two techniques were coupled with energy dispersive X-ray spectrometry for elemental analysis to complement wet geochemical data collected on bulk spring water and mat pore water. In the anoxic source of the spring, green and purple sulfur bacteria were found together with a sulfide-utilizing type of cyanobacteria that had the unusual characteristic of storing colloidal sulfur intracellularly. Deeper within the source, the mats were dominated by green sulfur bacteria and thick biofilms of cells that precipitated Fe and Zn sulfide minerals on their surfaces. Downstream from the source, thick, filamentous white mats lined the stream channel, formed by a diverse mass of nonphotosynthetic sulfur oxidizers, which were responsible for forming thick masses of spherical colloidal sulfur. These were distinguished by ESEM-EDS from cells by their simple elemental composition (only S was detected). Aqueous geochemistry analysis by ICP-MS showed that some elements (Fe, C, P, Zn, Mg, Ba) were present at higher levels in mat pore water than in bulk spring water. Our approach allowed us to gain an appreciation of the characteristics of this microbial community and allowed us to develop a good understanding of the types of microorganisms present and infer some of the relationships among the members of the community. In addition, we wish to convey the utility of a thorough microscopical approach in geomicrobiological and microbial ecology studies.     

Intact Phospholipid and Quinone Biomarkers to Assess Microbial Diversity and Redox State in Microbial Mats

Microbial mats are stratified microbial communities composed by highly inter-related populations and therefore are frequently chosen as model systems to study diversity and ecophysiological strategies. The present study describes an integrated approach to analyze microbial quinones and intact polar lipids (IPLs) in microbial mats within layers as thin as 500 μm by liquid chromatography–tandem mass spectrometry. Quinone profiles revealed important depth-related differences in community composition in two mat systems. The higher abundance of ubiquinones, compared to menaquinones, reflected the clear predominance of microorganisms belonging to aerobic α-, β-, and γ-Proteobacteria in Ebro delta estuarine mats. Hypersaline photosynthetic Camargue mats (France) showed a predominance of menaquinone-9 at the top of the mat, which is consistent with an important contribution of facultative aerobic or anaerobic bacteria in its photic zone. Quinone indices also indicated a higher diversity of non-phototrophs and a more anaerobic character in the hypersaline mats. Besides, the dissimilarity index suggested that the samples were greatly influenced by a depth-related redox state gradient. In the analysis of IPLs, there was a predominance of phosphatidylglycerols and sulfoquinovosyldiacylglycerols, the latter being an abundant biomarker of Cyanobacteria. This combined approach based on quinone and IPL analysis has proven to be a useful method to establish differences in the microbial diversity and redox state of highly structure microbial mat systems at a fine-scale level.     

Confocal Laser Scanning Microscopy Image Analysis for Cyanobacterial Biomass Determined at Microscale Level in Different Microbial Mats

We recently published a new method based on determining cyanobacterial biomass by confocal laser scanning microscopy image analysis (CLSM-IA) (Solé et al., Ultramicrosc 107:669–673, 2007). CLSM-IA allows biomass calculation for microorganisms of a small size, since the limit of the technique’s resolution is that generated by a voxel, the smallest unit of a three-dimensional digital image, equivalent to 1.183 × 10⁻³ mgC/cm³ of sediment. This method is especially suitable for the quantitative analysis of a large number of CLSM images generated from benthic sediments in which complex populations of cyanobacteria are abundant, such as microbial mats. In order to validate the new CLSM approach, mats with varying structural characteristics were studied. We have grouped them into three types: Microcoleus mats (laminated), sandy mats (nonlaminated and composed of well-sorted quartz sands), and oil-polluted mats. In this work, we applied CLSM-IA in natural [the Ebro delta and Sant Jordi colony (Spain), Salins-de-Giraud and Etang de Berre (France), and Orkney Islands (Scotland)] and artificial [mesocosms (Israel)] microbial mats. A total of 4,103 confocal images were obtained in order to determine total and individual cyanobacteria biomass profiles, at microscale level. The data presented in this paper show the efficacy of the method, as it can be applied to highly diverse mat samples.     

Jelly-like Microbial Mats over Subsurface Fields of Gas Hydrates at the St. Petersburg Methane Seep (Central Baikal)

Jelly-like microbial mat samples were collected from benthic surfaces at the St. Petersburg methane seep located in Central Baikal. The concentrations of certain ions, specifically chloride, bromide, sulphate, acetate, iron, calcium, and magnesium, were 2–40 times higher in the microbial mats than those in the pore and bottom water. A large number of diatom valves, cyanobacteria, and filamentous, rod-shaped and coccal microorganisms were found in the samples of bacterial mats using light, epifluorescence and scanning microscopy.Comparative analysis of a 16S rRNA gene fragment demonstrated the presence of bacteria and archaea belonging to the following classes and phyla: Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia, Cytophaga-Flavobacteria-Bacteroidetes, Cyanobacteria, and Euryarchaeota. The chemical composition and phylogenetic structure of the microbial community showed that the life activity of the mat occurs due to methane and its derivatives involved. Values of δ¹³C for the microbial mats varied from ?73.6‰ to ?65.8‰ and for animals from ?68.9‰ to ?36.6‰. Functional genes of the sequential methane oxidation (pmoA and mxaF) and different species of methanotrophic bacteria inhabiting cold ecosystems were recorded in the total DNA. Like in other psychroactive communities, the destruction of organic substances forming formed as a result of methanotrophy, terminates at the stage of acetate formation in the microbial mats of Lake Baikal (1,400 m depth). Its further transformation is limited by hydrogen content and carried out in the subsurface layers of sediments.     

Microstructural Characterization of Cyanobacterial Mats from the McMurdo Ice Shelf, Antarctica

The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.     

Microstructural characterization of cyanobacterial mats from the McMurdo Ice Shelf, Antarctica

The three-dimensional structures of two types of cyanobacterium-dominated microbial mats from meltwater ponds on the McMurdo Ice Shelf were as determined by using a broad suite of complementary techniques, including optical and fluorescence microscopy, confocal scanning laser microscopy, scanning electron microscopy with back-scattered electron-imaging mode, low-temperature scanning electron microscopy, and microanalyitical X-ray energy dispersive spectroscopy. By using a combination of the different in situ microscopic techniques, the Antarctic microbial mats were found to be structures with vertical stratification of groups of cyanobacteria and mineral sediments, high contents of extracellular polymeric substances, and large void spaces occupied by water. In cyanobacterium-rich layers, heterocystous nostocalean and nonheterocystous oscillatorialean taxa were the most abundant taxa and appeared to be intermixed with fine-size deposits of epicellular silica and calcium carbonate. Most of the cyanobacterial filaments had similar orientations in zones without sediment particles, but thin filaments were tangled among thicker filaments. The combination of the microscopic techniques used showed the relative positions of biological and mineral entities within the microbial mats and enabled some speculation about their interactions.     

FluoroNanogold is a bifunctional immunoprobe for correlative fluorescence and electron microscopy.

We applied a fluorescent ultrasmall immunogold probe, FluoroNanogold (FNG), to immunocytochemistry on ultrathin cryosections. FNG has the properties of both a fluorescent dye-conjugated antibody for fluorescence microscopy and a gold particle-conjugated antibody for electron microscopy. Therefore, this bifunctional immunoprobe permits correlative microscopic observation of the same cell profiles labeled in a single labeling procedure by these two imaging methods. We demonstrate the utility of FNG as a secondary antibody for immunocytochemical labeling of myeloperoxidase (a marker protein for azurophilic granules) in ultrathin cryosectioned human neutrophils. Its detection requires high spatial resolution because neutrophils contain many cytoplasmic granules. There was a one-to-one relationship between fluorescent structures labeled with FNG and organelle profiles labeled with the same silver-enhanced FNG in ultrathin cryosections. Use of FNG immunocytochemistry on ultrathin cryosections is an ideal methodology for high-resolution correlative fluorescence and electron microscopy and can provide unique information that may be difficult to obtain with a single imaging regimen.     

Structure of a microbiol mat at Great Sippewissett Marsh, Cape Cod, Massachusetts

The microzination of phototrophic bacteria in a flat laminated microbiol mat at Great Sippewissett Salt Marsh on Cape Cod, Massachusetts, was studied using a combination of scanning and transmission electron microscopy, light microscopy and photosynthetic pigment analysis. Comparison of pigment content and ultrastructural information from electron microscopy of thin sections allowed us to determine the major groups of photosynthetic bacteria present. The approximately 1-cm-thick mat is located in sandy intertidal sediments of the marsh and comprised four to five distinctly colored layers. The uppermost brown layer contained Lyngbya, Nostoc, Phormidium (cyanobacteria) and Navicula (diatom) species. An intermediate bluish-green layer was dominated by Oscillatoria species. A central pink layer contained purple sulfur bacteria such as Amoebobacter, Thiocapsa, Chromatium and Thiocystis species, Below this was a distinctive orange layer, formed largely by one species of purple sulfur bacteria, Thiocapsa pfennigii. The lowermost and thinnest layer contained green sulfur bacteria of the genus Prosthecochloris, a very small prosthecate species with numerous knobby projections; this layer was not always present. Below this, where pigments were generally absent, were dark gray and black iron sulfide-rich sediments. Remnants of older decayed mats could be found deeper in the sediment. Extensive production of microbial extracellular polymers in all layers appeared to be responsible for attachment of cells to sand grains, for lamination of layers and for structural integrity of the mat as a whole. Below the layer of green sulfur bacteria, binding of sediment by microbial polymers ceased abruptly. Possibly in response to decreasing light penetration, the mean size of bacterial cells decreased in successively deeper layers. In the lowest layer where light penetration was very low, green sulfur bacteria with highly convoluted surfaces occurred. The increase in cell surface area-to-volume ratio may allow such organisms to survive at low light levels.     

Community structure of free-floating filamentous cyanobacterial mats from the Wonder Lake geothermal springs in the Philippines

Cyanobacterial mats were characterized from pools of 45-60 degrees C in near-neutral pH, low-sulphide geothermal springs in the Philippines. Mat structure did not vary with temperature. All mats possessed highly ordered layers of airspaces at both the macroscopic and microscopic level, and these appear to be an adaptation to a free-floating growth habit. Upper mat layers supported biomass with elevated carotenoid:chlorophyll a ratios and an as yet uncharacterized waxy layer on the dorsal surface. Microscopic examination revealed mats comprised a single Fischerella morphotype, with abundant heterocysts throughout mats at all temperatures. Molecular analysis of mat community structure only partly matched morphological identification. All samples supported greater 16S rDNA-defined diversity than morphology suggested, with a progressive loss in the number of genotypes with increasing temperature. Fischerella-like sequences were recovered from mats occurring at all temperatures, but some mats also yielded Oscillatoria-like sequences, although corresponding phenotypes were not observed. Phylogenetic analysis revealed that Fischerella-like sequences were most closely affiliated with Fischerella major and the Oscillatoria-like sequences with Oscillatoria amphigranulata.     

Diazotrophic microbial community of coastal microbial mats of the southern North Sea

The diazotrophic community in microbial mats growing along the shore of the North Sea barrier island Schiermonnikoog (The Netherlands) was studied using microscopy, lipid biomarkers, stable carbon (δ(13) C(TOC) ) and nitrogen (δ(15) N) isotopes as well as by constructing and analyzing 16S rRNA gene libraries. Depending on their position on the littoral gradient, two types of mats were identified, which showed distinct differences regarding the structure, development and composition of the microbial community. Intertidal microbial mats showed a low species diversity with filamentous non-heterocystous Cyanobacteria providing the main mat structure. In contrast, supratidal microbial mats showed a distinct vertical zonation and a high degree of species diversity. Morphotypes of non-heterocystous Cyanobacteria were recognized as the main structural component in these mats. In addition, unicellular Cyanobacteria were frequently observed, whereas filamentous heterocystous Cyanobacteria occurred only in low numbers. Besides the apparent visual dominance of cyanobacterial morphotpyes, 16S rRNA gene libraries indicated that both microbial mat types also included members of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides group as well as diatoms. Bulk δ(15) N isotopes of the microbial mats ranged from +6.1‰ in the lower intertidal to -1.2‰ in the supratidal zone, indicating a shift from predominantly nitrate utilization to nitrogen fixation along the littoral gradient. This conclusion was supported by the presence of heterocyst glycolipids, representing lipid biomarkers for nitrogen-fixing heterocystous Cyanobacteria, in supratidal but not in intertidal microbial mats. The availability of combined nitrogen species might thus be a key factor in controlling and regulating the distribution of the diazotrophic microbial community of Schiermonnikoog.     

Unusual microbial mat‐related structural diversity 2.1 billion years ago and implications for the Francevillian biota

The 2.1‐billion‐year‐old (Ga) Francevillian series in Gabon hosts some of the oldest reported macroscopic fossils of various sizes and shapes, stimulating new debates on the origin, evolution and organization of early complex life. Here, we document ten representative types of exceptionally well‐preserved mat‐related structures, comprising “elephant‐skin” textures, putative macro‐tufted microbial mats, domal buildups, flat pyritized structures, discoidal microbial colonies, horizontal mat growth patterns, wrinkle structures, “kinneyia” structures, linear patterns and nodule‐like structures. A combination of petrographic analyses, scanning electron microscopy, Raman spectroscopy and organic elemental analyses of carbon‐rich laminae and microtexture, indicate a biological origin for these structures. The observed microtextures encompass oriented grains, floating silt‐sized quartz grains, concentrated heavy minerals, randomly oriented clays, wavy‐crinkly laminae and pyritized structures. Based on comparisons with modern analogues, as well as an average δ¹³C organic matter (C_org) composition of ?32.94 ± 1.17‰ (1 standard deviation, SD) with an outlier of ?41.26‰, we argue that the mat‐related structures contain relicts of multiple carbon pathways including heterotrophic recycling of photosynthetically derived C_org. Moreover, the relatively close association of the macroscopic fossil assemblages to the microbial mats may imply that microbial communities acted as potential benthic O₂ oases linked to oxyphototrophic cyanobacterial mats and grazing grounds. In addition, the mat's presence likely improved the preservation of the oldest large colonial organisms, as they are known to strongly biostabilize sediments. Our findings highlight the oldest community assemblage of microscopic and macroscopic biota in the aftermath of the “Great Oxidation Event,” widening our understanding of biological organization during Earth's middle age.     

Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C₁ compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.     

Comparative microbial diversity analyses of modern marine thrombolitic mats by barcoded pyrosequencing

Thrombolites are unlaminated carbonate structures that form as a result of the metabolic interactions of complex microbial mat communities. Thrombolites have a long geological history; however, little is known regarding the microbes associated with modern structures. In this study, we use a barcoded 16S rRNA gene-pyrosequencing approach coupled with morphological analysis to assess the bacterial, cyanobacterial and archaeal diversity associated with actively forming thrombolites found in Highborne Cay, Bahamas. Analyses revealed four distinct microbial mat communities referred to as black, beige, pink and button mats on the surfaces of the thrombolites. At a coarse phylogenetic resolution, the domain bacterial sequence libraries from the four mats were similar, with Proteobacteria and Cyanobacteria being the most abundant. At the finer resolution of the rRNA gene sequences, significant differences in community structure were observed, with dramatically different cyanobacterial communities. Of the four mat types, the button mats contained the highest diversity of Cyanobacteria, and were dominated by two sequence clusters with high similarity to the genus Dichothrix, an organism associated with the deposition of carbonate. Archaeal diversity was low, but varied in all mat types, and the archaeal community was predominately composed of members of the Thaumarchaeota and Euryarchaeota. The morphological and genetic data support the hypothesis that the four mat types are distinctive thrombolitic mat communities.     

Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.     

The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities

We have examined the biosynthesis and accumulation of cyanobacterial sunscreening pigment scytonemin within intertidal microbial mat communities using a combination of chemical, molecular, and phylogenetic approaches. Both laminated (layered) and nonlaminated mats contained scytonemin, with morphologically distinct mats having different cyanobacterial community compositions. Within laminated microbial mats, regions with and without scytonemin had different dominant oxygenic phototrophs, with scytonemin-producing areas consisting primarily of Lyngbya aestuarii and scytonemin-deficient areas dominated by a eukaryotic alga. The nonlaminated mat was populated by a diverse group of cyanobacteria and did not contain algae. The amplification and phylogenetic assignment of scytonemin biosynthetic gene scyC from laminated mat samples confirmed that the dominant cyanobacterium in these areas, L. aestuarii, is likely responsible for sunscreen production. This study is the first to utilize an understanding of the molecular basis of scytonemin assembly to explore its synthesis and function within natural microbial communities.     

Biogeochemistry and community composition of iron- and sulfur-precipitating microbial mats at the Chefren mud volcano (Nile Deep Sea Fan, Eastern Mediterranean)

In this study we determined the composition and biogeochemistry of novel, brightly colored, white and orange microbial mats at the surface of a brine seep at the outer rim of the Chefren mud volcano. These mats were interspersed with one another, but their underlying sediment biogeochemistries differed considerably. Microscopy revealed that the white mats were granules composed of elemental S filaments, similar to those produced by the sulfide-oxidizing epsilonproteobacterium "Candidatus Arcobacter sulfidicus." Fluorescence in situ hybridization indicated that microorganisms targeted by a "Ca. Arcobacter sulfidicus"-specific oligonucleotide probe constituted up to 24% of the total the cells within these mats. Several 16S rRNA gene sequences from organisms closely related to "Ca. Arcobacter sulfidicus" were identified. In contrast, the orange mat consisted mostly of bright orange flakes composed of empty Fe(III) (hydr)oxide-coated microbial sheaths, similar to those produced by the neutrophilic Fe(II)-oxidizing betaproteobacterium Leptothrix ochracea. None of the 16S rRNA gene sequences obtained from these samples were closely related to sequences of known neutrophilic aerobic Fe(II)-oxidizing bacteria. The sediments below both types of mats showed relatively high sulfate reduction rates (300 nmol x cm(-3) x day(-1)) partially fueled by the anaerobic oxidation of methane (10 to 20 nmol x cm(-3) x day(-1)). Free sulfide produced below the white mat was depleted by sulfide oxidation within the mat itself. Below the orange mat free Fe(II) reached the surface layer and was depleted in part by microbial Fe(II) oxidation. Both mats and the sediments underneath them hosted very diverse microbial communities and contained mineral precipitates, most likely due to differences in fluid flow patterns.     

A precise and rapid mapping protocol for correlative light and electron microscopy of small invertebrate organisms

Background information. CLEM (correlative live cell and electron microscopy) seeks to bridge the data acquired with different imaging strategies, typically between light microscopy and electron microscopy. It has been successfully applied in cell cultures, although its use in multicellular systems is hampered by difficulties in locating the ROI (region of interest). Results. We developed a CLEM technique that enables easy processing of small model animals and is adequate both for morphology and immunoelectron‐microscopic specimen preparations. While this method has been initially developed for Caenorhabditis elegans samples, we found that it works equally well for Drosophila samples. It enables handling and observation of single animals of any complex genotype in real time, fixation by high‐pressure freezing and flat embedding. Our major improvement has been the development of a precise mapping system that considerably simplifies and speeds up the retrospective location of the ROI within 1 μm distance. This method can be successfully used when correlative microscopy is required, as well as to facilitate the treatment of non‐correlative TEM procedures. Our improvements open the possibility to treat statistically significant numbers of animals processed by electron microscopy and considerably simplifies electron‐microscopic protocols, making them more accessible to a wider range of researchers. Conclusions. We believe that this technique will contribute to correlative studies in multicellular models and will facilitate the time‐demanding procedure of specimen preparation for any kind of TEM.