Biogeochemical cycling and microbial diversity in the thrombolitic microbialites of Highborne Cay,Bahamas

Thrombolites are unlaminated carbonate build‐ups that are formed via the metabolic activities of complex microbial mat communities. The thrombolitic mats of Highborne Cay, Bahamas develop in close proximity (1–2 m) to accreting laminated stromatolites, providing an ideal opportunity for biogeochemical and molecular comparisons of these two distinctive microbialite ecosystems. In this study, we provide the first comprehensive characterization of the biogeochemical activities and microbial diversity of the Highborne Cay thrombolitic mats. Morphological and molecular analyses reveal two dominant mat types associated with the thrombolite deposits, both of which are dominated by bacteria from the taxa Cyanobacteria and Alphaproteobacteria. Diel cycling of dissolved oxygen (DO) and dissolved inorganic carbon (DIC) were measured in all thrombolitic mat types. DO production varied between thrombolitic types and one morphotype, referred to in this study as ‘button mats’, produced the highest levels among all mat types, including the adjacent stromatolites. Characterization of thrombolite bacterial communities revealed a high bacterial diversity, roughly equivalent to that of the nearby stromatolites, and a low eukaryotic diversity. Extensive phylogenetic overlap between thrombolitic and stromatolitic microbial communities was observed, although thrombolite‐specific cyanobacterial populations were detected. In particular, the button mats were dominated by a calcified, filamentous cyanobacterium identified via morphology and 16S rRNA gene sequencing as Dichothrix sp. The distinctive microbial communities and chemical cycling patterns within the thrombolitic mats provide novel insight into the biogeochemical processes related to the lithifying mats in this system, and provide data relevant to understanding microbially induced carbonate biomineralization.     

Microbial diversity in modern marine stromatolites, Highborne Cay, Bahamas

Living marine stromatolites at Highborne Cay, Bahamas, are formed by microbial mat communities that facilitate precipitation of calcium carbonate and bind and trap small carbonate sand grains. This process results in a laminated structure similar to the layering observed in ancient stromatolites. In the modern marine system at Highborne Cay, lamination, lithification and stromatolite formation are associated with cycling between three types of microbial communities at the stromatolite surface (Types 1, 2 and 3, which range from a leathery microbial mat to microbially fused sediment). Examination of 923 universal small-subunit rRNA gene sequences from these communities reveals that taxonomic richness increases during transition from Type 1 to Type 3 communities, supporting a previous model that proposed that the three communities represent different stages of mat development. The phylogenetic composition also changes significantly between these community types and these community changes occur in concert with variation in biogeochemical rates. The dominant bacterial groups detected in the stromatolites include Alphaproteobacteria , Planctomycetes , Cyanobacteria and Bacteroidetes . In addition, the stromatolite communities were found to contain novel cyanobacteria that may be uniquely associated with modern marine stromatolites. The implications of these findings are discussed in the context of current models for stromatolite formation.     

Active eukaryotes in microbialites from Highborne Cay,Bahamas, and Hamelin Pool (Shark Bay), Australia

Microbialites are organosedimentary structures that are formed through the interaction of benthic microbial communities and sediments and include mineral precipitation. These lithifying microbial mat structures include stromatolites and thrombolites. Exuma Sound in the Bahamas, and Hamelin Pool in Shark Bay, Western Australia, are two locations where significant stands of modern microbialites exist. Although prokaryotic diversity in these structures is reasonably well documented, little is known about the eukaryotic component of these communities and their potential to influence sedimentary fabrics through grazing, binding and burrowing activities. Accordingly, comparisons of eukaryotic communities in modern stromatolitic and thrombolitic mats can potentially provide insight into the coexistence of both laminated and clotted mat structures in close proximity to one another. Here we examine this possibility by comparing eukaryotic diversity based on Sanger and high-throughput pyrosequencing of small subunit ribosomal RNA (18S rRNA) genes. Analyses were based on total RNA extracts as template to minimize input from inactive or deceased organisms. Results identified diverse eukaryotic communities particularly stramenopiles, Alveolata, Metazoa, Amoebozoa and Rhizaria within different mat types at both locations, as well as abundant and diverse signatures of eukaryotes with <80% sequence similarity to sequences in GenBank. This suggests the presence of significant novel eukaryotic diversity, particularly in hypersaline Hamelin Pool. There was evidence of vertical structuring of protist populations and foraminiferal diversity was highest in bioturbated/clotted thrombolite mats of Highborne Cay.     

Molecular biosignatures reveal common benthic microbial sources of organic matter in ooids and grapestones from Pigeon Cay,The Bahamas

Ooids are sedimentary grains that are distributed widely in the geologic record. Their formation is still actively debated, which limits our understanding of the significance and meaning of these grains in Earth's history. Central questions include the role played by microbes in the formation of ooids and the sources of ubiquitous organic matter within ooid cortices. To address these issues, we investigated the microbial community composition and associated lipids in modern oolitic sands at Pigeon Cay on Cat Island, The Bahamas. Surface samples were taken along a transect from the shallow, turbulent surf zone to calmer, deeper water. Grains transitioned from shiny and abraded ooids in the surf zone, to biofilm‐coated ooids at about 3 m water depth. Further offshore, grapestones (cemented aggregates of ooids) dominated. Benthic diatoms and Proteobacteria dominated biofilms. Taxa that may promote carbonate precipitation were abundant, particularly those associated with sulfur cycling. Compared to the lipids associated with surface biofilms, relict lipids bound within carbonate exhibited remarkably similar profiles in all grain types. The enhanced abundance of methyl‐branched fatty acids and β‐hydroxy fatty acids, 1‐O‐monoalkyl glycerol ethers and hopanoids bound within ooid and grapestone carbonate confirms a clear association of benthic sedimentary bacteria with these grains. Lipids bound within ooid cortices also contain molecular indicators of microbial heterotrophic degradation of organic matter, possibly in locally reducing conditions. These included the loss of labile unsaturated fatty acids, enhanced long‐chain fatty acids/short‐chain fatty acids, enriched stable carbon isotopes ratios of fatty acids, and very high stanol/stenol ratios. To what extent some of these molecular signals are derived from later heterotrophic endolithic activity remains to be fully resolved. We speculate that some ooid carbonate forms in microbial biofilms and that early diagenetic degradation of biofilms may also play a role in early stage carbonate precipitation around ooids.     

Functional gene diversity of oolitic sands from Great Bahama Bank

Despite the importance of oolitic depositional systems as indicators of climate and reservoirs of inorganic C, little is known about the microbial functional diversity, structure, composition, and potential metabolic processes leading to precipitation of carbonates. To fill this gap, we assess the metabolic gene carriage and extracellular polymeric substance (EPS) development in microbial communities associated with oolitic carbonate sediments from the Bahamas Archipelago. Oolitic sediments ranging from high‐energy ‘active’ to lower energy ‘non‐active’ and ‘microbially stabilized’ environments were examined as they represent contrasting depositional settings, mostly influenced by tidal flows and wave‐generated currents. Functional gene analysis, which employed a microarray‐based gene technology, detected a total of 12 432 of 95 847 distinct gene probes, including a large number of metabolic processes previously linked to mineral precipitation. Among these, gene‐encoding enzymes for denitrification, sulfate reduction, ammonification, and oxygenic/anoxygenic photosynthesis were abundant. In addition, a broad diversity of genes was related to organic carbon degradation, and N₂ fixation implying these communities has metabolic plasticity that enables survival under oligotrophic conditions. Differences in functional genes were detected among the environments, with higher diversity associated with non‐active and microbially stabilized environments in comparison with the active environment. EPS showed a gradient increase from active to microbially stabilized communities, and when combined with functional gene analysis, which revealed genes encoding EPS‐degrading enzymes (chitinases, glucoamylase, amylases), supports a putative role of EPS‐mediated microbial calcium carbonate precipitation. We propose that carbonate precipitation in marine oolitic biofilms is spatially and temporally controlled by a complex consortium of microbes with diverse physiologies, including photosynthesizers, heterotrophs, denitrifiers, sulfate reducers, and ammonifiers.     

Molecular community analysis of microbial diversity

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.     

Molecular microbial diversity of a spacecraft assembly facility

In ongoing investigations to map and archive the microbial footprints in various components of the spacecraft and its accessories, we have examined the microbial populations of the Jet Propulsion Laboratory's Spacecraft Assembly Facility (JPL-SAF). Witness plates made up of spacecraft materials, some painted with spacecraft qualified paints, were exposed for 7 to 9 months at JPL-SAF and examined the particulate materials collected for the incidence of total cultivable aerobic heterotrophs and heat-tolerant (80°C for 15-min.) spore-formers. The results showed that the witness plates coated with spacecraft qualified paints attracted more dust particles than the non-coated stainless steel witness plates. Among the four paints tested, witness plates coated with NS43G accumulated the highest number of particles, and hence attracted more cultivable microbes. The conventional microbiological examination revealed that the JPL-SAF harbors mainly Gram-positive microbes and mostly spore-forming Bacillus species. Most of the isolated microbes were heat resistant to 80°C and proliferate at 60°C. The phylogenetic relationships among 23 cultivable heat-tolerant microbes were examined using a battery of morphological, physiological, molecular and chemotaxonomic characterizations. By 16S rDNA sequence analysis, the isolates fell into seven clades:Bacillus licheniformis, B. pumilus, B. cereus, B. circulans, Staphylococcus capitis, Planococcus sp. and Micrococcus lylae. In contrast to the cultivable approach, direct DNA isolation, cloning and 16S rDNA sequencing analysis revealed equal representation of both Gram-positive and Gram-negative microorganisms.     

Molecular analysis of microbial diversity in the Zavarzin Spring,Uzon Caldera,Kamchatka

The Zavarzin spring is situated in the caldera of the Uzon volcano, Kamchatka, and is characterized by a temperature of about 60°C, neutral pH, and high concentration of sulfur. The bottom of the spring is covered with a cyanobacterial mat. The structure of the microbial community of the water from the Zavarzin spring was qualitatively and quantitatively characterized by pyrosequencing of the V3 variable region of the 16S rRNA gene, which yielded 37 654 independent sequences. The microbial community includes about 900 bacterial and 90 archaeal genera. Bacteria comprised 95% of the microorganisms and archaea less than 5%. The largest part (32.3%) of the community was constituted by the chemolithoautotrophic bacteria Aquificae from the genera Sulfurihydrogenibium and Thermosulfidibacter. Among autotrophic microorganisms, members of Thermodesulfobacteria (7.3%), the gammaproteobacteria Thiofaba (7.6%), the deltaproteobacteria Desulfurella (2.6%), and the betaproteobacteria Thiomonas (0.6%) were also identified. Heterotrophic bacteria were represented by Calditerrivibrio (12.1%), Thermotogae (6.3%), the betaproteobacteria Tepidimonas (6.0%), Deinococcus-Thermus (4.4%), Caldiserica (1.7%), and Dictyoglomi (1.6%). About 1.9% of microorganisms belonged to the BRC1 phylum, which does not include cultured members, and 0.2% of bacteria formed a new phylogenetic branch of the phylum level, representatives of which have been found only in the Zavarzin spring. Members of all four archaeal phyla were identified: Euryarchaeota (42% of archaeal sequences), Crenarchaeota (50%), Korarchaeota (7.5%), and Nanoarchaeota (0.5%). Thus, in the Zavarzin spring, apart from photosynthesis carried out by the cyanobacterial mat, which covers the bottom, chemolithoautotrophic production of organic matter can occur. In aerobic conditions, it proceeds at the expense of the oxidation of sulfur and its reduced compounds, and in anaerobic conditions, at the expense of the oxidation of hydrogen with sulfur and sulfates as electron acceptors. The organic matter formed by autotrophic bacteria may be utilized by various organotrophic microorganisms, including both fermentative bacteria and organisms that carry out anaerobic respiration with sulfur and nitrate as electron acceptors.     

Bifidobacterial diversity and the development of new microbial source tracking indicators

Many studies suggest a close relationship between species of Bifidobacterium and their hosts. Thus, species such as B. adolescentis and B. thermacidophilum subsp. porcinum have been proposed as potential indicators of human and porcine fecal pollution. The diversity of bifidobacteria in wastewaters (human and animal) and slurries is analyzed using nested PCR followed by denaturant gradient gel electrophoresis (DGGE). The sewage samples showed similar DGGE patterns. The predominant bands were recognized as B. adolescentis, B. longum, and two unidentified species related to B. adolescentis. A single band detected in poultry samples was identified as B. saeculare. Bifidobacterial diversity was higher within porcine and bovine samples. The main bands in porcine samples were identified as B. minimum, an unknown species, and B. thermophilum/B. thermacidophilum subsp. porcinum. The latter species was also identified among the main bands in bovine samples together with B. pseudolongum and B. ruminantium. We then attempted to isolate the host-specific strains. DGGE bands were examined to develop specific probes to screen environmental samples by colony hybridization and further isolation of strains from positively hybridized colonies. Bifidobacterial strains that are host associated by DGGE bands to human and pig were successfully isolated from the environment: B. adolescentis from human sewage samples and the unidentified species related to pig from slurries and slaughterhouse wastewater. Neither the poultry-associated B. saeculare nor the ruminant-associated B. pseudolongum could be isolated with the current methodology, suggesting either a low prevalence in the samples or failure of the culture to grow in the media used.     

Molecular microbial diversity of an agricultural soil in Wisconsin.

A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA). A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed. Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil. The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced. The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined. Of the 124 clones sequenced, 98.4% were from the domain Bacteria. Two of the rDNA sequences were derived from eukaryotic organelles. Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence. No sequences of the domain Archaea were found. Within the domain, Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low G+C-content gram-positive group (21.8%). Some kingdoms, such as the Thermotogales, the green nonsulfur group, Fusobacteria, and the Spirochaetes, were absent. A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al. (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol., 176:1-6, 1994). From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.     

Ecophysiology of stromatolitic microbial mats,Stocking Island,exuma cays,Bahamas

Intertidal stromatolites, covered by cyanobacterial mats, were recently discovered at Stocking Island, Exuma Cays, Bahamas. Ecophysiological responses (CO₂ fixation, N₂ fixation, and photoacclimation) of these cyanobacterial mats to experimental manipulations were examined to identify potential environmental variables controlling community structure and function. The mats exhibit horizontal zonation that shifts from soft to crusty to hard in a seaward direction. Cluster analysis of chemotaxonomic photopigments (chlorophylls and carotenoids) revealed that visually distinct mat types are composed of distinct phototrophic assemblages. Under reduced irradiance, diatoms within the mats photoacclimated by increasing accessory photopigments (diadinoxanthin, fucoxanthin, and chlorophyll c ₁ c ₂) and cyanobacteria reduced the photoprotective carotenoid echinenone. In a 4-day nutrient addition bioassay experiment, nitrate, phosphate, dissolved organic carbon, and trace metal enrichments did not enhance CO₂ fixation, but phosphate enrichments tripled N₂ fixation rates. The addition of DCMU increased N₂ fixation rates relative to nonamended light and dark rates, indicating light (photosystem I) enhanced nitrogenase activity. Soft mats appear to represent the early stages of colonization and stabilization of mat communities. Active growth following stabilization results in the formation of partially-lithified crusty mats, which eventually become highly-lithified and form hard mats. Collectively, our results suggest that Stocking Island stromatolitic mats have low growth rates and consequently exhibit slow responses to increased nutrient availability and changes in ambient irradiance. In general, intertidal stromatolitic mats at Stocking Island appear to exhibit low rates of CO₂ and N₂ fixation relative to nonlithifying temperate cyanobacteral mats. Although production is low, respiration is likewise low, leading to the suggestion that high production to respiration ratios (P:R) may be necessary for lithification of intertidal stromatolitic mats.     

Molecular evaluation of microbial diversity occurring in different types of Mozzarella cheese

AIMS: The microbial community of different types of unripened Pasta Filata cheese was investigated by culture-independent methods with the aim of rapidly achieving knowledge about cheese microbiota and discriminating traditional and industrial cheeses. METHODS AND RESULTS: The microbial DNA extracted directly from the samples was used as a template in PCR experiments to amplify the 16S-23S rDNA spacer region and the V3 region of the 16S rDNA. Conventional electrophoresis of the amplified spacers allowed known classes of these DNA fragments belonging to genera and species of lactic acid bacteria to be distinguished. Denaturing gradient gel electrophoresis analysis of V3 amplicons was supported by reference cultures of LAB used as markers. CONCLUSION: Both molecular approaches furnished the expected information about microbial diversity and were quite valid for discriminating industrial, semi-artisanal or traditional cheeses, characterized by increasingly complex DNA profiles. SIGNIFICANCE AND IMPACT OF THE STUDY: Both methods could be used for legal purposes when products obtained through prescribed manufacturing regulations are to be analysed.     

Molecular diversity of microbial eukaryotes in sea water from Fildes Peninsula,King George Island,Antarctica

Data on Antarctic coastal sites remain scarce and are generally limited to microscopy; the diversity of coastal Antarctic ecosystems has long been underestimated. The diatom-dominant community in the coastal sea waters of the Fildes Peninsula has been described according to traditional protocols. Molecular diversity of microbial eukaryotes (≤20 µm) from Great Wall Cove and Ardley Cove, Fildes Peninsula, has been determined by Illumina MiSeq2000 sequencing. Inferred metabolisms of summer phytoplankton in the two coves are characterised by autotrophy and heterotrophy. The frequent occurrence of such nanoflagellates as dinoflagellates, Cryptophyta, Stramenopiles, Pyramimonas, Telonema, and Cryothecomonas is predicted to be important in these Antarctic coastal communities. Sea water exchange exists between the two coves when high tide occurs, indicating that there appears to be mixing between the microbial communities in the two coves. Cluster analysis of the microbial eukaryote composition at the phylum and genus levels reveals a conservation of the community composition between the two coves. The inner stations of Great Wall Cove represented by three shoal samples (G1, G2, and G3) are clustered closely together, surrounded by islands and formed into a semi-closed body of water. The samples from the outer stations G4 and G5 of Great Wall Cove, which are separated from the other three Great Wall stations, are more similar to the Ardley Cove samples. Sea water exchange between the outer basins might be the effect of their community compositions. The nanoplankton diversity in Great Wall Cove is richer than in Ardley Cove, according to the α-diversity index.     

Molecular analysis of microbial diversity in corrosion samples from energy transmission towers

Microbial diversity in corrosion samples from energy transmission towers was investigated using molecular methods. Ribosomal DNA fragments were used to assemble gene libraries. Sequence analysis indicated 10 bacterial genera within the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. In the two libraries generated from corroded screw-derived samples, the genus Acinetobacter was the most abundant. Acinetobacter and Clostridium spp. dominated, with similar percentages, in the libraries derived from corrosion scrapings. Fungal clones were affiliated with 14 genera belonging to the phyla Ascomycota and Basidiomycota; of these, Capnobotryella and Fellomyces were the most abundant fungi observed. Several of the microorganisms had not previously been associated with biofilms and corrosion, reinforcing the need to use molecular techniques to achieve a more comprehensive assessment of microbial diversity in environmental samples.     

Molecular analysis of microbial diversity in corrosion samples from energy transmission towers

Microbial diversity in corrosion samples from energy transmission towers was investigated using molecular methods. Ribosomal DNA fragments were used to assemble gene libraries. Sequence analysis indicated 10 bacterial genera within the phyla Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. In the two libraries generated from corroded screw-derived samples, the genus Acinetobacter was the most abundant. Acinetobacter and Clostridium spp. dominated, with similar percentages, in the libraries derived from corrosion scrapings. Fungal clones were affiliated with 14 genera belonging to the phyla Ascomycota and Basidiomycota; of these, Capnobotryella and Fellomyces were the most abundant fungi observed. Several of the microorganisms had not previously been associated with biofilms and corrosion, reinforcing the need to use molecular techniques to achieve a more comprehensive assessment of microbial diversity in environmental samples.     

Molecular indicators of microbial contributions to recent and Tertiary hypersaline lacustrine sediments in China

In this paper, we investigate the molecular characteristics of recent sediment from the Ejinur salt lake (northern China) and eight Tertiary (Eocene) core samples from Qianjiang Formation (Eq1-4) of Jianghan basin, central eastern China, focussing on the distributions and abundances of carboxylic acid isolated from extractable organic matter. n-Alkanoic acids in sediments from both study areas (Ejinur and Jianghan) show a pronounced even over odd predominance (EOP) and a bimodel distribution. In the lower molecular weight range C16 and C_18 components are prominent with the former dominant. For higher homologues (≥ C20), docosanoic (C22) and tetracosanoic (C24) acids dominant the n-alkanoic acid homologues for the Jianghan and Ejinur samples, respectively. Alkanoic acids with an isoprenoid skeleton are more abundant in Jianghan, including C20, C21, C24, C_25 and C30 homologues, with a C25 component (3, 7, 11, 15, 19-pentamethyleicosanoic acid) most pronounced in the lower part of the Qianjiang Fm. The carbon skeletons of these isoprenoid acids are identical to those of isoprenoid hydrocarbons previously recognized in source rocks and petroleums from Jianghan, and attributed to Archaea (formally called archaebacteria; e.g. halophiles, methanogens). The similarity in the distributions of isoprenoid alkanoic acids and alkanes suggests that these isoprenoid acids must derive from comparable biological sources, although they appear to be formed geochemically from biosynthetic precursors (e.g. alkanes, alkenes, alkanols or alkenols). The possibility that these acids are formed as oxidation artifacts from alkanes can be discounted because of the dissimilarity between the distributions of n-alkanoic acids and n-alkanes. Iso and anteiso branched carboxylic acids are prevalent in both the Ejinur sample and the upper portion of the Qiangiang Fm (Eq1-3). They derive from bacteria, perhaps sulfate reducing bacteria, and their abundances may aid assessment of the importance of bacterial contributions of organic matter in different sedimentary environments. The presence of hopanoid acids and a 3-carboxy steroidal acid further attest to contributions from bacterial and eukaryotic sources, respectively. The occurrence of carboxylic acids in the Jianghan samples confirms the potential for these compounds to survive in ancient sediments and source rocks, notably in hypersaline settings. This revised version was published online in July 2006 with corrections to the Cover Date.     

分子生物学方法在微生物多样性研究中的应用

微生物多样性是生物多样性的重要组成部分。由于微生物和大生物（动、植物）相比,存在着多种显著差异,因此其多样性,保护及利用也有所不同,尤其是研究方法亟待完善,提高。近年来,分子生物学方法广泛用于微生物多样性的研究并取得了一系列研究成果。本文从四个方面加以介绍：１）微生物总ＤＮＡ制备及其遗传多样性检测方法;２）１６ＳｒＲＮＡ基因序列研究;３）核酸杂交分析技术;４）ＤＮＡ动力学的研究。今后的发展趋势是加