Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) sulfidic springs dominated by chemolithoautotrophic "Epsilonproteobacteria"

Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave, Wyoming, were assessed with regard to bacterial diversity, community structure, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and stable carbon isotope ratio analyses. The most prevalent mat morphotype consisted of white filament bundles, with low C:N ratios (3.5-5.4) and high sulfur content (16.1-51.2%). White filament bundles and two other mat morphotypes had organic carbon isotope values (mean delta13C=-34.7 per thousand, 1sigma=3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean delta13C=-7.4 per thousand for two springs, n=8). Bacterial diversity was low overall in the clone libraries, and the most abundant taxonomic group was affiliated with the "Epsilonproteobacteria" (68%), with other bacterial sequences affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Six distinct epsilonproteobacterial taxonomic groups were identified from the microbial mats. Epsilonproteobacterial and bacterial group abundances and community structure shifted from the spring orifices downstream, corresponding to changes in dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the clone sequences for epsilonproteobacterial groups were retrieved from areas with high sulfide and low oxygen concentrations, whereas Thiothrix spp. and Thiobacillus spp. had higher retrieved clone abundances where conditions of low sulfide and high oxygen concentrations were measured. Genetic and metabolic diversity among the "Epsilonproteobacteria" maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Our results demonstrate that sulfur cycling supports subsurface ecosystems through chemolithoautotrophy and expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats.     

Occurrence of photosynthetic microbial mats in a Lower Cretaceous black shale (central Italy): a shallow-water deposit

Cretaceous oceanic anoxic events (OAEs) were periods of high organic carbon burial corresponding to intervals with excellent organic matter (OM) preservation. This work focuses on the Urbino level, i.e., OAE1b, which is thought to be of regional extent. A detailed microscopical study of OM shows a dominance of microbial activity, characterized by a typical arrangement of exopolymeric substances (EPS) related to microbial mats, bacterial bodies, and some photosynthetic microorganisms, as shown by thylakoids. The latter lived where they have been found, i.e., at the sea bottom, which indicates that OM results from the diagenesis of benthic photosynthetic microbial mats, an interpretation supported through the comparison with a recent analogue. The exceptional preservation of such organic structures in OM points to the joint role of the selective and sorptive preservation pathways. These data and interpretation strongly differ from previous observations in OAE1b equivalents. They suggest that the Urbino level might be an atypical OAE of regional/local extent which was formed within the photic zone.     

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.     

Melanosclerites from the Wilhelmi Formation (Lower Silurian,north-eastern Illinois,USA)

Melanosclerites, a little-known group of organic microfossils, are recorded for the first time from the Lower Silurian Wilhelmi Formation, Illinois, USA. The taxa described herein include Melanorhachis regularis Eisenack, 1942, Melanoarbustum balticum Górka 1971, Melanocyathus dentatus Eisenack, 1942, Eichbaumia incus? Schallreuter, 1981, Menola os Schallreuter, 1981 and Melanoporella clava Schallreuter, 1981.     

Characterization, seasonal occurrence, and diel fluctuation of poly(hydroxyalkanoate) in photosynthetic microbial mats

In situ poly(hydroxyalkanoate) (PHA) levels and repeating-unit compositions were examined in stratified photosynthetic microbial mats from Great Sippewissett Salt Marsh, Mass., and Ebro Delta, Spain. Unlike what has been observed in pure cultures of phototrophic bacteria, the prevalence of hydroxyvalerate (HV) repeating units relative to hydroxybutyrate (HB) repeating units was striking. In the cyanobacteria-dominated green material of Sippewissett mats, the mole percent ratio of repeating units was generally 1HB:1HV. In the purple sulfur bacteria-dominated pink material the relationship was typically 1HB:2HV. In Sippewissett mats, PHA contributed about 0.5 to 1% of the organic carbon in the green layer and up to 6% in the pink layer. In Ebro Delta mats, PHA of approximately 1HB:2HV-repeating-unit distribution contributed about 2% of the organic carbon of the composite photosynthetic layers (the green and pink layers were not separated). Great Sippewissett Salt Marsh mats were utilized for more extensive investigation of seasonal, diel, and exogenous carbon effects. When the total PHA content was normalized to organic carbon, there was little seasonal variation in PHA levels. However, routine daily variation was evident at all sites and seasons. In every case, PHA levels increased during the night and decreased during the day. This phenomenon was conspicuous in the pink layer, where PHA levels doubled overnight. The daytime declines could be inhibited by artificial shading. Addition of exogenous acetate, lactate, and propionate induced two- to fivefold increases in the total PHA levels when applied in the daylight but had no effect when applied at night. The distinct diel pattern of in situ PHA accumulation at night appears to be related, in some phototrophs, to routine dark energy metabolism and is not influenced by the availability of organic nutrients.     

In situ analysis of oxygen consumption and diffusive transport in high‐temperature acidic iron‐oxide microbial mats

The role of dissolved oxygen as a principal electron acceptor for microbial metabolism was investigated within Fe(III)‐oxide microbial mats that form in acidic geothermal springs of Yellowstone National Park (USA). Specific goals of the study were to measure and model dissolved oxygen profiles within high‐temperature (65–75°C) acidic (pH = 2.7–3.8) Fe(III)‐oxide microbial mats, and correlate the abundance of aerobic, iron‐oxidizing Metallosphaera yellowstonensis organisms and mRNA gene expression levels to Fe(II)‐oxidizing habitats shown to consume oxygen. In situ oxygen microprofiles were obtained perpendicular to the direction of convective flow across the aqueous phase/Fe(III)‐oxide microbial mat interface using oxygen microsensors. Dissolved oxygen concentrations dropped from ～ 50–60 μM in the bulk‐fluid/mat surface to below detection (< 0.3 μM) at a depth of ～ 700 μm (～ 10% of the total mat depth). Net areal oxygen fluxes into the microbial mats were estimated to range from 1.4–1.6 × 10^?4 μmol cm^?2 s^?1. Dimensionless parameters were used to model dissolved oxygen profiles and establish that mass transfer rates limit the oxygen consumption. A zone of higher dissolved oxygen at the mat surface promotes Fe(III)‐oxide biomineralization, which was supported using molecular analysis of Metallosphaera yellowstonensis 16S rRNA gene copy numbers and mRNA expression of haem Cu oxidases (FoxA) associated with Fe(II)‐oxidation.     

Spatial heterogeneity of eukaryotic microbial communities in an unstudied geothermal diatomaceous biological soil crust: Yellowstone National Park, WY, USA

Knowledge of microbial communities and their inherent heterogeneity has dramatically increased with the widespread use of high-throughput sequencing technologies, and we are learning more about the ecological processes that structure microbial communities across a wide range of environments, as well as the relative scales of importance for describing bacterial communities in natural systems. Little work has been carried out to assess fine-scale eukaryotic microbial heterogeneity in soils. Here, we present findings from a bar-coded 18S rRNA survey of the eukaryotic microbial communities in a previously unstudied geothermal diatomaceous biological soil crust in Yellowstone National Park, WY, USA, in which we explicitly compare microbial community heterogeneity at the particle scale within soil cores. Multivariate analysis of community composition showed that while subsamples from within the same soil core clustered together, community dissimilarity between particles in the same core was high. This study describes a novel soil microbial environment and also adds to our growing understanding of microbial heterogeneity and the scales relevant to the study of soil microbial communities.     

Identification and characterization of Rhodopseudomonas spp., a purple, non-sulfur bacterium from microbial mats

A species of facultative photo-organotrophic, purple, non-sulfur bacterium was isolated from mixed-species microbial mats, characterized and examined for metal tolerance and bioremediation potential. Contributing mats were natural consortia of microbes, dominated by cyanobacteria and containing several species of bacteria arranged in a laminar structure, stabilized within a gel matrix. Constructed microbial mats were used for bioremediation of heavy metals and organic chemical pollutants. Purple, non-sulfur bacteria are characteristically found in lower strata of intact mats, but their contributing function in mats survival and function by mediating the chemical environment has not been explored. The gram-negative rod-shaped bacterium, reported here, produced a dark red culture under phototrophic conditions, reproduced by budding and formed a lamellar intracytoplasmic membrane (ICM) system parallel to cytoplasmic membrane, which contained bacteriochlorophyll a and carotenoids. This strain was found to have multiple metal resistances and to be effective in the reductive removal of Cr(VI) and the degradation of 2,4,6-trichlorophenol. Based on the results obtained from morphology, nutrient requirements, major bacteriochlorophyll content, GC content, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) profile and 16S-rDNA phylogenetic analysis, this member of the microbial mats may be identified as a new strain of the genus Rhodopseudomonas.     

Protozoan communities of the Flint River-Lake Blackshear ecosystem (Georgia,USA)

Freshwater protozoa are poorly characterized in river ecosystems. We report here the richness of the protozoan biotas in relation to environmental gradients from an ecosystematic survey of a large, coastal plain river. Communities were collected from natural and artificial substrates concurrent with water chemistry analysis at 11 sites along the Flint River and Lake Blackshear impoundment. Community similarity, the distribution of collected taxa in functional feeding groups, and the relation of communities to environmental gradients were evaluated. Two principal compenents determined from water chemistry data showed important downstream gradients of decreasing water hardness and increasing nutrient levels. Taxonomic richness was high; 200 to 450 taxa were collected depending on season and collecting technique. Artificial substrates provided the richest collections. Bactivorous species were the vast majority of all taxa collected. Community composition showed an orderly transition from upstream to downstream, and photosynthetic forms were enhanced at nutrient enriched sites. Communities were strongly influenced by increasing nutrient levels. Protozoan community analysis showed that microbial community composition reflects human influences on river ecosystems. Since microbial species exploit detrital resources and respond sensitively to human influences, they can provide important information regarding ecosystem conditions.     

Genetic diversity patterns of microbial communities in a subtropical riverine ecosystem (Jiulong River,southeast China)

Prokaryotic and eukaryotic microbes are key organisms in aquatic ecosystems and play pivotal roles in the biogeochemical cycles, but little is known about genetic diversity of these communities in subtropical rivers. In this study, microbial planktonic communities were determined by using denaturing gradient gel electrophoresis (DGGE) analysis from the Jiulong River, southeast China, and their relationships with local environmental factors were studied. The Betaproteobacteria (26%) and Dinophyceae (26%) were the most dominant taxa in prokaryotic and eukaryotic clones derived from DGGE bands, respectively. Further, both cluster and ordination analyses of prokaryotic and eukaryotic DGGE fingerprinting resulted in three identical groups from the 15 sites, which were closely related with the environmental factors. Partial redundancy analysis (partial RDA) revealed that agricultural pollution (phosphorus and nitrogen) and saltwater intrusion (conductivity and salinity) were the main factors impacting microbial community composition, by explaining more than two-thirds of the total variation in both prokaryotic (67.0%) and eukaryotic (70.5%) communities. Moreover, the robust and quantifiable relationship between DGGE results and environmental variables indicated that the community-level molecular fingerprinting techniques could support the physicochemical assessment of riverine water quality and ecosystem health.     

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min.     

In situ ecological development of a bacteriogenic iron oxide-producing microbial community from a subsurface granitic rock environment

The initial development and diversity of an in situ subsurface microbial community producing bacteriogenic iron oxides (BIOS) were investigated at the initiation of biofilm growth (2‐month period) and after a 1‐year period of undisturbed growth. Water chemistry data, samples of iron encrusted biofilm material and groundwater were collected from BRIC (BIOS reactor, in situ, continuous flow) apparatuses installed 297 m below sea level at the Äspö Hard Rock Laboratory (HRL) in south eastern Sweden. Comparisons between the BIOS BRIC system and an anaerobic control (AC) BRIC revealed that water mixing at the inflow leads to profuse development of BIOS related to a slightly elevated level of O₂ (up to 0.3 mg L^?1 at the transition zone between BIOS development and non‐development) and elevated Eh (>120 mV) in the first 70 mm of water depth. Decreases in dissolved and particulate iron were connected to the visible appearance of BIOS biofilms. The basic phylogenetic diversity of this site was evaluated using amplified ribosomal DNA restriction enzyme analysis (ARDRA), denaturing gradient gel electrophoresis (DGGE) and partial sequencing of 16S rDNA. From 67 clones that were positive for 16S rDNA inserts, a total of 42 different ARDRA profiles were recognized, representing four bacterial phyla and 14 different metabolic lifestyles. DGGE profiles indicated that there are differences in the representative bacteria when considering either BIOS biofilms or groundwater. DGGE also indicated that the DNA extraction protocols and any polymerase chain reaction biases were consistent. Bacterial metabolic groups associated with indirect metal adsorption and reduction along with bacteria utilizing many alternative electron acceptors were strongly represented within the clones. This study indicates that the microbial diversity of BIOS is greater than previously thought.     

In situ visualization of high genetic diversity in a natural microbial community.

Simultaneous in situ visualization of seven distinct bacterial genotypes, all affiliated with the phylogenetically narrow group of beta-1 Proteobacteria, was achieved in activated sludge. This finding indicates that the high diversity found in the same sample by direct rRNA sequence retrieval was indeed present in this complex community. By the combination of comparative rRNA sequence analysis, in situ hybridization with fluorescently labeled, rRNA-targeted oligonucleotides and confocal laser scanning microscopy several microbial populations can be analyzed for abundance, relative spatial distribution and phylogeny directly at their site of action without prior cultivation.     

Diversity of inorganic carbon acquisition mechanisms by intact microbial mats of Microcoleus chthonoplastes (Cyanobacteriae, Oscillatoriaceae)

The dissolved inorganic carbon (DIC) acquisition mechanisms were researched in intact microbial mats dominated by the cyanobacteria Microcoleus chthonoplastes Thuret, by determining the effect on photosynthesis of different inhibitors. The microbial mats exhibited high affinity for DIC at alkaline pH, with K(m(DIC)) values similar to the ones described for pure cultures of cyanobacteria and algae in which carbon concentrating mechanisms have been researched. Besides, the photosynthesis was non-sensitive to pH changes within the range of 5.6-9.6, indicating that HCO(3)(-) was the main DIC source used for photosynthesis. The M. chthonoplastes mats featured external and internal carbonic anhydrase (CA) activity as measured in intact cells and cell extracts, respectively. Acetazolamide (AZ, which slowly enters the cell and then inhibits mainly the external CA) and ethoxyzolamide (EZ, which inhibits both external and internal CA) reduced significantly the oxygen evolution rates, demonstrating that the CA was implied in the DIC acquisition. Vanadate inhibited photosynthesis by 60% although its application, when CA being inhibited (i.e. after applying AZ + EZ), did not produce any additional effect. It could indicate that ATPase-dependent HCO(3)(-) use occurred and also that this putative mechanism was coupled with CA-like activity at the plasma membrane. The involvement of Na(+)-dependent HCO(3)(-) transporters in DIC acquisition was also inferred as monensin and 4-4'-diisothiocyanatostibilene-2,2'-disulfonate (DIDS) reduced photosynthesis by 70%. DIDS produced a strong inhibitory effect even after application of AZ + EZ + vanadate, indicating that this mechanism was not related to CA activity. The microbial mats become subject to very unfavourable conditions for Rubisco carboxylation at their natural habitats (e.g. external pH of 10.5 and O(2) concentration doubled with respect to saturation concentration); therefore, this putative diversity of DIC acquisition mechanisms could ensure their growth under these extreme conditions.     

Molecular diversity studies of bacterial communities of oil polluted microbial mats from the Etang de Berre (France)

The biodiversity of microbial mats inhabiting the oil-contaminated lagoon Etang de Berre was determined by molecular approaches. The fingerprint of denaturing gradient gel electrophoresis (DGGE) and automatic ribosomal intergenic spacer analysis (ARISA) of mats exposed to different pollution levels showed specific microbial communities for each site but similar diversity richness. Species composition of the mats were compared by constructing 16S rRNA libraries. Amplified rDNA restriction analysis (ARDRA) of clone libraries confirmed their similar level of diversity richness. Phylogenetic analysis of the 16S rRNA sequences showed that the classes gamma and alpha of Proteobacteria were abundantly present in both sites whereas phylotypes related to the delta-Proteobacteria and to the uncultured WS3 group were mainly found in the site with the highest pollution. Identification of the species involved in oil degradation by combining culture-based approaches and DGGE, showed that enrichment cultures were constituted by members of the Rhodobacterales and species related to Rhodococcus, Sphingomonas, Xanthomonas and Microbacterium, all of them known for their ability to degrade hydrocarbons. Our findings suggest that oil pollution has not affected the biodiversity richness of the mats. However, the populations involved in hydrocarbon degradation represent a minor fraction of the mat communities in the Etang de Berre.     

Nobloedischia rasnitsyni, a new genus and species of Oedischiidae (Orthoptera) from the Lower Permian Wellington Formation of Oklahoma, USA

Nobloedischia rasnitsynigen. et sp. n. (Oedischiidae) is described from the Lower Permian Wellington Formation of Noble County, Oklahoma. The genus is similar to both Petrelcana (Oedischiidae: Mezenoedischiinae) and Oedischia (Oedischiidae: Oedischiinae) and is left unplaced at the subfamily level. The new species is the twelfth Orthoptera species and the fourth species of Oedischiidae from these deposits.     

Community structure and physiological characterization of microbial mats in Byers Peninsula, Livingston Island (South Shetland Islands, Antarctica)

The community structure and physiological characteristics of three microbial mat communities in Byers Peninsula (Livingston Island, South Shetland Islands, Antarctica) were compared. One of the mats was located at the edge of a stream and was dominated by diatoms (with a thin basal layer of oscillatorian cyanobacteria), whereas the other two mats, located over moist soil and the bottom of a pond, respectively, were dominated by cyanobacteria throughout their vertical profiles. The predominant xanthophyll was fucoxanthin in the stream mat and myxoxanthophyll in the cyanobacteria-dominated mats. The sheath pigment scytonemin was absent in the stream mat but present in the soil and pond mats. The stream mat showed significantly lower delta13C and higher delta15N values than the other two mats. Consistent with the delta15N values, N2 fixation was negligible in the stream mat. The soil mat was the physiologically most active community. It showed rates of photosynthesis three times higher than in the other mats, and had the highest rates of ammonium uptake, nitrate uptake and N2 fixation. These observations underscore the taxonomic and physiological diversity of microbial mat communities in the maritime Antarctic region.     

Role of the microbial community in formation of speleothem (moonmilk) in the Snezhnaya carst cave (abkhazia)

The results of investigation of speleothem (moonmilk) from the Snezhnaya cave (West Caucasus, Abkhazia) are reported. The structure of microbial complexes from moonmilk was investigated by quantitative PCR; strains of culturable microorganisms were isolated and their preferred temperature and carbon sources were determined. Among eubacteria, ~34% belonged to the iron bacteria (Rhodoferax). Most bacterial strains were shown to be facultative psychrophiles with the maximum growth rate at 4°C. The microstructure and elemental composition of moonmilk were investigated using a scanning electron microscope (EVO-40HV, Carl Zeiss) and silicon drift X-ray detector X-MAX 80mm². The visually plastic and homogeneous mass of moonmilk was shown to be highly heterogeneous, containing various microstructures. The elemental composition of some nanostructures depended on the structure of bacterial biofilms. Some loci of the biofilm were found to contain up to 46% (wt/wt) of iron oxides. High calcium content, up to 61.5% (wt/wt), was found only in cubic crystalline structures which were not involved in microbiological processes.