Chloroflexus spp. and Sulfurihydrogenibium spp. in microbial mats of the Nakabusa hot spring, Japan

Microbial mats develop in a wide range of aquatic habitats, such as geothermal hot springs, hypersaline ponds, marine cold seeps or hydrothermal vents. The Nakabusa hot spring is located in the Nagano Prefecture, Japan (36.3875N, 137.75E), dense olive-green microbial mats develop in regions where the slightly alkaline, sulfidic effluent has cooled to 65°C. The microbial community of such mats was analyzed by focusing on the diversity, as well as the in situ distribution and function of bacteria involved in sulfur cycling. Microbial mat samples were kept in sterile plastic tubes (for molecular analysis) or glass bottles completely filled with hot spring water to avoid oxidation. Samples were transferred to the laboratory on ice and used for physiological experiments within 8h. Quantification of cell biovolumes was carried out based on images of mat sections hybridized with Sulfurihydrogenibium- and Chloroflexi-specific probes, and stained with DAPI. In situ hybridizations (CARD-FISH) of thin matsections showed a heterogeneous vertical distribution of Sulfurihydrogenibium and Chloroflexus. Sulfurihydrogenibium dominated near the mat surface (50% of the total mat biovolume), while Chloroflexus dominated in deeper layers (up to 64% of the total mat biovolume).

Microbial phyllosphere populations are more complex than previously realized

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97–100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists

The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.

Molecular investigation of the microbial populations of the pink sugarcane mealybug, Saccharicoccus sacchari

In an attempt to better understand the microbial diversity and endosymbiotic microbiota of the pink sugarcane mealybug (PSMB) Saccharicoccus sacchari Cockerell (Homoptera: Pseudococcidae), culture-independent approaches, namely PCR, a 16S rDNA clone library, and temperature gradient gel electrophoresis (TGGE) were used. Previous work has indicated that the acetic acid bacteria Gluconacetobacter sacchari, Gluconacetobacter diazotrophicus, and Gluconacetobacter liquefaciens represent only a small proportion of the microbial community of the PSMB. These findings were supported in this study by TGGE, where no bands representing G. sacchari, G. diazotrophicus, and G. liquefaciens on the acrylamide gel could be observed following electrophoresis, and by a 16S rDNA clone library study, where no clones with the sequence of an acetic acid bacterium were found. Instead, TGGE revealed that the mealybug microbial community was dominated by beta- and gamma-Proteobacteria. The dominant band in TGGE gels found in a majority of the mealybug samples was most similar, according to BLAST analysis, to the beta-symbiont of the craw mealybug Antonina crawii and to Candidatus Tremblaya princeps, an endosymbiont from the mealybug Paracoccus nothofagicola. The sequences of other dominant bands were identified as gamma-Proteobacteria, and were most closely related to uncultured bacterial clones obtained from soil samples. Mealybugs collected from different areas in Queensland, Australia, were found to produce similar TGGE profiles, although there were a few exceptions. A 16S rDNA clone library based on DNA extracted from a mealybug collected from sugarcane in the Burdekin region in Queensland, Australia, indicated very low levels of diversity among mealybug microbial populations. All sequenced clones were most closely related to the same members of the gamma-Proteobacteria, according to BLAST analysis.

Challenges for simultaneous nitrification, denitrification, and phosphorus removal in microbial aggregates: mass transfer limitation and nitrous oxide production

The microbial community composition and activity was investigated in aggregates from a lab-scale bioreactor, in which nitrification, denitrification and phosphorus removal occurred simultaneously. The biomass was highly enriched for polyphosphate accumulating organisms facilitating complete removal of phosphorus from the bulk liquid; however, some inorganic nitrogen still remained at the end of the reactor cycle. This was ascribed to incomplete coupling of nitrification and denitrification causing NO3- accumulation. After 2 h of aeration, denitrification was dependent on the activity of nitrifying bacteria facilitating the formation of anoxic zones in the aggregates; hence, denitrification could not occur without simultaneous nitrification towards the end of the reactor cycle. Nitrous oxide was identified as a product of denitrification, when based on stored PHA as carbon source. This observation is of critical importance to the outlook of applying PHA-driven denitrification in activated sludge processes. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Structure of a cellulose degrading bacterial community during anaerobic digestion

It is widely accepted that cellulose is the rate-limiting substrate in the anaerobic digestion of organic solid wastes and that cellulose solubilisation is largely mediated by surface attached bacteria. However, little is known about the identity or the ecophysiology of cellulolytic microorganisms from landfills and anaerobic digesters. The aim of this study was to investigate an enriched cellulolytic microbial community from an anaerobic batch reactor. Chemical oxygen demand balancing was used to calculate the cellulose solubilisation rate and the degree of cellulose solubilisation. Fluorescence in situ hybridisation (FISH) was used to assess the relative abundance and physical location of three groups of bacteria belonging to the Clostridium lineage of the Firmicutes that have been implicated as the dominant cellulose degraders in this system. Quantitation of the relative abundance using FISH showed that there were changes in the microbial community structure throughout the digestion. However, comparison of these results to the process data reveals that these changes had no impact on the cellulose solubilisation in the reactor. The rate of cellulose solubilisation was approximately stable for much of the digestion despite changes in the cellulolytic population. The solubilisation rate appears to be most strongly affected by the rate of surface area colonisation and the biofilm architecture with the accepted model of first order kinetics due to surface area limitation applying only when the cellulose particles are fully covered with a thin layer of cells. (c) 2005 Wiley Periodicals, Inc.

Microbial diversity within the water column of a larval rearing system for the ornate rock lobster (Panulirus ornatus)

The ornate tropical rock lobster, Panulirus ornatus has substantial potential as an aquaculture species though disease outbreaks during the animal's extended larval lifecycle are major constraints for success. In order to effectively address such disease-related issues, an improved understanding of the composition and dynamics of the microbial communities in the larval rearing tanks is required. This study used flow cytometry and molecular microbial techniques (clone libraries and denaturing gradient gel electrophoresis (DGGE)) to quantify and characterise the microbial community of the water column in the early stages (developmental stage I-II) of a P. ornatus larval rearing system. DGGE analysis of a 5000 L larval rearing trial demonstrated a dynamic microbial community with distinct changes in the community structure after initial stocking (day I to day 2) and from day 4 to day 5, after which the structure was relatively stable. Flow cytometry analysis of water samples taken over the duration of the trial demonstrated a major increase in bacterial load leading up to and peaking on the first day of the initial larval moult (day 7), before markedly decreasing prior to when > 50% of larvae moulted (day 9). A clone library of a day 10 water sample taken following a mass larval mortality event reflected high microbial diversity confirmed by statistical analysis indices. Sequences retrieved from both clone library and DGGE analyses were dominated by gamma- and alpha-Proteobacteria affiliated organisms with additional sequences affiliated with beta- and epsilon-Proteobacteria, Bacteroidetes, Cytophagales and Chlamydiales groups. Vibrio affiliated species were commonly retrieved in the clone library, though absent from DGGE analysis.

Relative significance of pelagic, benthic and allochthonous organic matter to microbial carbon cycling in a phosphorus-depleted subtropical estuary (Florida Bay)

Heterotrophic bacteria are important decomposers and transformers of primary production and provide an important link between detritus and the aquatic food web. In seagrass ecosystems, much of seagrass primary production is unavailable through direct grazing and must undergo microbial reworking before seagrass production can enter the aquatic food web. The goal of my dissertation research is to understand better the role heterotrophic bacteria play in carbon cycling in seagrass estuaries. My dissertation research focuses on Florida Bay, a seagrass estuary that has experienced recent changes in carbon source availability, which may have altered ecosystem function. My dissertation research investigates the importance of seagrass, algal and/or cyanobacterial, and allochthonous-derived organic matter to heterotrophic bacteria in Florida Bay and helps establish the carbon base of the estuarine food web. ^ A three tiered approach to the study of heterotrophic bacterial carbon cycling and trophic influences in Florida Bay was used: (1) Spatiotemporal observations of environmental parameters (hydrology, nutrients, extracellular enzymes, and microbial abundance, biomass, and production); (2) Microbial grazing experiments under different levels of top-down and bottom-up influence; and (3) Bulk and compound-specific (bacteria-biomarker fatty acid analysis) stable carbon isotope analysis. ^ In Florida Bay, spatiotemporal patterns in microbial extracellular enzyme (also called ectoenzyme) activities indicate that microorganisms hydrolyzed selectively fractions of the estuarine organic matter pool. The microbial community hydrolyzed organic acids, peptides, and phosphate esters and did not use storage and structural carbohydrates. Organic matter use by heterotrophic bacterioplankton in Florida Bay was co-regulated by bottom-up (resource availability) and top-down (grazer mediated) processes. A bacterial carbon budget based on bacterial, epiphytic, and seagrass production indicates that heterotrophic bacterial carbon cycles are supported primarily through epiphytic production with mixing from seagrass production. Stable carbon isotope analysis of bacteria biomarkers and carbon sources in Florida Bay corroborate the results of the bacterial carbon budget. These results support previous studies of aquatic consumers in Florida Bay, indicating that epiphytic/benthic algal and/or cyanobacterial production with mixing from seagrass-derived organic matter is the carbon base of the seagrass estuarine food web. ^

Patterns of Soil Bacteria and Canopy Community Structure Related to Tropical Peatland Development

Natural environmental gradients provide important information about the ecological constraints on plant and microbial community structure. In a tropical peatland of Panama, we investigated community structure (forest canopy and soil bacteria) and microbial community function (soil enzyme activities and respiration) along an ecosystem development gradient that coincided with a natural P gradient. Highly structured plant and bacterial communities that correlated with gradients in phosphorus status and soil organic matter content characterized the peatland. A secondary gradient in soil porewater NH4 described significant variance in soil microbial respiration and β-1-4-glucosidase activity. Covariation of canopy and soil bacteria taxa contributed to a better understanding of ecological classifications for biotic communities with applicability for tropical peatland ecosystems of Central America. Moreover, plants and soils, linked primarily through increasing P deficiency, influenced strong patterning of plant and bacterial community structure related to the development of this tropical peatland ecosystem.

Sediment microbial communities at CO2 seeps in Papua New Guinea

To understand how ocean acidification (OA) influences sediment microbial communities, naturally CO2-rich sites are increasingly being used as OA analogues. However, the characterization of these naturally CO2-rich sites is often limited to OA-related variables, neglecting additional environmental variables that may confound OA effects. Here, we used an extensive array of sediment and bottom water parameters to evaluate pH effects on sediment microbial communities at hydrothermal CO2 seeps in Papua New Guinea. The geochemical composition of the sediment pore water showed variations in the hydrothermal signature at seep sites with comparable pH, allowing the identification of sites that may better represent future OA scenarios. At these sites, we detected a 60% shift in the microbial community composition compared with reference sites, mostly related to increases in Chloroflexi sequences. pH was among the factors significantly, yet not mainly, explaining changes in microbial community composition. pH variation may therefore often not be the primary cause of microbial changes when sampling is done along complex environmental gradients. Thus, we recommend an ecosystem approach when assessing OA effects on sediment microbial communities under natural conditions. This will enable a more reliable quantification of OA effects via a reduction of potential confounding effects. This pangaea entry contains the data on the microbial community structure and bottom water parameters.

Soil properties and microbial indicators of samples from Lake Wellman, Darwin Mountains, Antarctica

Four pedons on each of four drift sheets in the Lake Wellman area of the Darwin Mountains were sampled for chemical and microbial analyses. The four drifts, Hatherton, Britannia, Danum, and Isca, ranged from early Holocene (10 ka) to mid-Quaternary (c. 900 ka). The soil properties of weathering stage, salt stage, and depths of staining, visible salts, ghosts, and coherence increase with drift age. The landforms contain primarily high-centred polygons with windblown snow in the troughs. The soils are dominantly complexes of Typic Haplorthels and Typic Haploturbels. The soils were dry and alkaline with low levels of organic carbon, nitrogen and phosphorus. Electrical conductivity was high accompanied by high levels of water soluble anions and cations (especially calcium and sulphate in older soils). Soil microbial biomass, measured as phospholipid fatty acids, and numbers of culturable heterotrophic microbes, were low, with highest levels detected in less developed soils from the Hatherton drift. The microbial community structure of the Hatherton soil also differed from that of the Britannia, Danum and Isca soils. Ordination revealed the soil microbial community structure was influenced by soil development and organic carbon.

Microbial metabolism mediates interactions between dissolved organic matter and clay minerals in streamwater

Sorption of organic molecules to mineral surfaces is an important control upon the aquatic carbon (C) cycle. Organo-mineral interactions are known to regulate the transport and burial of C within inland waters, yet the mechanisms that underlie these processes are poorly constrained. Streamwater contains a complex and dynamic mix of dissolved organic compounds that coexists with a range of organic and inorganic particles and microorganisms. To test how microbial metabolism and organo-mineral complexation alter amino acid and organic carbon fluxes we experimented with 13C-labelled amino acids and two common clay minerals (kaolinite and montmorillonite). The addition of 13C-labelled amino acids stimulated increased microbial activity. Amino acids were preferentially mineralized by the microbial community, concomitant with the leaching of other (non-labelled) dissolved organic molecules that were removed from solution by clay-mediated processes. We propose that microbial processes mediate the formation of organo-mineral particles in streamwater, with potential implications for the biochemical composition of organic matter transported through and buried within fluvial environments.

Quantification of microbial communities in subsurface marine sediments of the Black Sea and off Namibia

Organic-rich subsurface marine sediments were taken by gravity coring up to a depth of 10 m below seafloor at six stations from the anoxic Black Sea and the Benguela upwelling system off Namibia during the research cruises Meteor 72-5 and 76-1, respectively. The quantitative microbial community composition at various sediment depths was analyzed using total cell counting, catalyzed reporter deposition fluorescence in situ hybridization (CARD FISH) and quantitative real-time PCR (Q-PCR). Total cell counts decreased with depths from 10(9) to 10(10) cells/mL at the sediment surface to 10(7)-10(9) cells/mL below one meter depth. Based on CARD FISH and Q-PCR analyses overall similar proportions of Bacteria and Archaea were found. The down-core distribution of prokaryotic and eukaryotic small subunit ribosomal RNA genes (16S and 18S rRNA) as well as functional genes involved in different biogeochemical processes was quantified using Q-PCR. Crenarchaeota and the bacterial candidate division JS-1 as well as the classes Anaerolineae and Caldilineae of the phylum Chloroflexi were highly abundant. Less abundant but detectable in most of the samples were Eukarya as well as the metal and sulfate-reducing Geobacteraceae (only in the Benguela upwelling influenced sediments). The functional genes cbbL, encoding for the large subunit of RuBisCO, the genes dsrA and aprA, indicative of sulfate-reducers as well as the mcrA gene of methanogens were detected in the Benguela upwelling and Black Sea sediments. Overall, the high organic carbon content of the sediments goes along with high cell counts and high gene copy numbers, as well as an equal abundance of Bacteria and Archaea.

Bacterial community characterization of water and intestine of the shrimp Litopenaeus stylirostris in a biofloc system

Background Biofloc technology (BFT), a rearing method with little or no water exchange, is gaining popularity in aquaculture. In the water column, such systems develop conglomerates of microbes, algae and protozoa, together with detritus and dead organic particles. The intensive microbial community presents in these systems can be used as a pond water quality treatment system, and the microbial protein can serve as a feed additive. The current problem with BFT is the difficulty of controlling its bacterial community composition for both optimal water quality and optimal shrimp health. The main objective of the present study was to investigate microbial diversity of samples obtained from different culture environments (Biofloc technology and clear seawater) as well as from the intestines of shrimp reared in both environments through high-throughput sequencing technology. Results Analyses of the bacterial community identified in water from BFT and “clear seawater” (CW) systems (control) containing the shrimp Litopenaeus stylirostris revealed large differences in the frequency distribution of operational taxonomic units (OTUs). Four out of the five most dominant bacterial communities were different in both culture methods. Bacteria found in great abundance in BFT have two principal characteristics: the need for an organic substrate or nitrogen sources to grow and the capacity to attach to surfaces and co-aggregate. A correlation was found between bacteria groups and physicochemical and biological parameters measured in rearing tanks. Moreover, rearing-water bacterial communities influenced the microbiota of shrimp. Indeed, the biofloc environment modified the shrimp intestine microbiota, as the low level (27 %) of similarity between intestinal bacterial communities from the two treatments. Conclusion This study provides the first information describing the complex biofloc microbial community, which can help to understand the environment-microbiota-host relationship in this rearing system.

Response of Core Microbial Consortia to Chronic Hydrocarbon Contaminations in Coastal Sediment Habitats

Traditionally, microbial surveys investigating the effect of chronic anthropogenic pressure such as polyaromatic hydrocarbons (PAHs) contaminations consider just the alpha and beta diversity and ignore the interactions among the different taxa forming the microbial community. Here, we investigated the ecological relationships between the three domains of life (i.e., Bacteria, Archaea, and Eukarya) using 454 pyrosequencing on the 16S rRNA and 18S rRNA genes from chronically impacted and pristine sediments, along the coasts of the Mediterranean Sea (Gulf of Lion, Vermillion coast, Corsica, Bizerte lagoon and Lebanon) and the French Atlantic Ocean (Bay of Biscay and English Channel). Our approach provided a robust ecological framework for the partition of the taxa abundance distribution into 859 core Operational taxonomic units (OTUs) and 6629 satellite OTUs. OTUs forming the core microbial community showed the highest sensitivity to changes in environmental and contaminant variations, with salinity, latitude, temperature, particle size distribution, total organic carbon (TOC) and PAH concentrations as main drivers of community assembly. The core communities were dominated by Gammaproteobacteria and Deltaproteobacteria for Bacteria, by Thaumarchaeota, Bathyarchaeota and Thermoplasmata for Archaea and Metazoa and Dinoflagellata for Eukarya. In order to find associations among microorganisms, we generated a co-occurrence network in which PAHs were found to impact significantly the potential predator – prey relationship in one microbial consortium composed of ciliates and Actinobacteria. Comparison of network topological properties between contaminated and non-contaminated samples showed substantial differences in the network structure and indicated a higher vulnerability to environmental perturbations in the contaminated sediments.