thursday 23 august ps24 – microbial life in extreme ...llebbe/abstracts/isme 14... · in order to...

Thursday 23 August

1 PS24 – Microbial Life in Extreme Environments

PS24 – Microbial Life in Extreme Environments

076A Diversity and function of fungal communities in biological soil crusts in Oman Raeid Abed*, Abdullah Al-Sadi, Muneera Al Shehi, Sheikha Al-Hinai, Michael Robinson Sultan Qaboos University, Oman

Biological soil crusts of arid and semiarid regions of the world are recognized as one of the least explored niches occupied by fungi. Cultivation and 454 pyrosequencing were employed to investigate free-living and lichen-forming fungal communities associated with crusts at two sites in theSultanate of Oman. The role of fungi in improving soil stability and hydrology was further studied. A total of 390 fungal strains were obtained and phylogenetic analysis placed the majority of the fungal strains within the Ascomycota phylum with the dominance of Dothideomycetes class and Pleosporales order. A total of 44 different genera and 101 different species were identified with the dominance of the genera: Alternaria, Phoma, Fusarium, Cochliobolus, Ulocladium, Cladosporium and Myrothecium. Using pyrosequencing, a total of 26,998 sequence reads were obtained with Ascomycota, Basidiomycota and Chytridiomycota encompassing >96% of the total sequences. The sequences of the phylum Ascomycota were distributed in the classes Dothideomycete, Sordariomycete, Eurotiomycete, Pezizomycete, Leotiomycetes, Lecanoromycetes and Lichinomycetes. Among the identified lichens were Placidium lacinulatum, Psora decipiens, Peccania fontqueriana, Stromatella bermudana, Verrucaria chiloensis, Pecania arizonica, Lempholemma polyanthes and Lichinella cribellifera. Although detected fungi confirmed earlier trends in fungal diversity in other deserts, quite a number of strains and sequences seems to be endemic for Oman and were not reported elsewhere. The presence of lichen in crusts improved their resistance to erosion and increased their water holding capacity. Non-crusted soil lost 4.5 to 9 times more mass than cyanobacteria or lichen crust, respectively. We conclude that desert crusts of Oman harbor a large diversity of fungal communities, which appears to be linked to crust types as well as specific desert ecoregions, and the presence of lichens in crusts seems to improve soil characteristics.

077A Effects of simazine and 2, 4-dichlorophenoxyacetic acid upon genetics and metabolic diversity in aerobic heterotrophic bacteria present in a pristine cold aquatic oligotrophic environments Paulina Aguayo*1, Felipe Contreras2, Paulina González2, Evelyn Hernández2, Carlos González2, Ricardo Barra3, Miguel Martínez2 1Universidad de Concepción, Microbiología, Chile, 2Departamento de Microbiología, Universidad de Concepción, Chile, 3Centro EULA-Chile, Universidad de Concepción, Chile

Pristine cold aquatic oligotrophic lakes as those found in the Chilean Patagonia have physical and chemical conditions that harbor different types of bacteria. However, these environments frequently show fluctuations in their physicochemical parameters, like temperature and nutrients offer. Témpanos Lake, located in the northern region of the Chilean Patagonia (Aysén, Chile), is an oligotrophic lake with temperatures under 10°C. In this pristine lake, it is possible to find microbial communities composed mainly by bacteria adapted to harsh conditions and able to maintain their viability in this ecosystem. Although this type of environment is characterized by low human intervention, it is expected an increase of industrial activity of the Aysén region in the near future which will expose the lake to increase their nutrient offer with the additions of organic matter, including toxic compound. This new carbon offer may induce change in the composition of the microbial communities or alter the metabolism and physiology of the resident bacterial communities. The aim of this work was to investigate the effects of simazine and 2,4-dichlorophenoxyacetic acid in microcosms of sediment from the Témpanos Lake and to assess its effect on the metabolic and genetic diversity of the bacterial communities of this oligotrophic lake. Viability was estimated as bacterial viable counts and live/dead proportion by epifluorescence microscopy. Genetic and metabolic diversity was analyzed in microcosm using superficial sediment, obtained in summer and winter, by using DGGE and Biolog Ecoplate system, respectively. The results showed a higher percentage of recoverable bacterial cells in summer (9,0%) as compared to winter samples (5,2%). There was no decrease in viable counts of the microcosms supplemented with simazine or 2,4-dichlorophenoxyacetic acid. The genetic profile of microcosm supplemented with simazine and 2,4-dichlorophenoxyacetic showed a larger number of OTUS with respect to the control in both seasons. In addition, during summer microcosms used wider Carbon sources (97% of the offer) as compared to winter samples (81%) but intensity of consume was lower. This behavior was observed in spite of the presence of Simazine or 2,4-dichlorophenoxyacetic.

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The results obtained suggest that incorporation of organic matter to pristine lakes might affect the intensity of use of substrates by resident bacterial communities. Easily metabolization substrates were the main carbon source affected by the season and this could explain changes in the genetic diversity found in this community.

078A Spatial and salinity effect in halophilic bacterial diversity of Salar de Huasco (Chilean Altiplano) Pablo Aguilar1, Cristina Dorador1, Joice Leon2, Francisco Remonsellez*2 1Universidad de Antofagasta, Chile, 2Universidad Catolica del Norte, Chile

Salar de Huasco is a saline wetland located at 3,800 meters altitude in the Chilean Altiplano; it is considered an athalassohaline system because its salt composition is markedly different from that of seawater. This saline system exhibits typical conditions of the Altiplano, including low temperatures (mean annual temperature < 5°C), low atmospheric pressure (40% lower than that at sea level), high solar radiation, strong daily variation in different environmental properties, negative water balance, and salinity conditions from freshwater to saturated salt waters; which could account for microbial communities inhabiting these water bodies.

For this study we collected samples from sites H3, H4 and H6, previously described, exhibiting salinity values of 0.3%, 12.3% and 1.2%, respectively. In order to study the bacterial diversity at different NaCl concentration, and to identify novel halophiles and halotolerant bacterial communities; we constructed clone libraries from PCR products of 16S rRNA gene. In total, 193 sequences from clone libraries were obtained from water samples, and only 2 sequences were 100% identical. Rarefaction curves from the three sites showed saturation with 20 phylotypes to H3, 35 phylotypes to H4, and 39 phylotypes to H6. Many of the all phylotypes identified (52%) exhibited similarities lower than 97% to their closest relatives analyzed in the Genbank. The sequences analysis showed the presence of 8, 13 and 16 phyla, for H3, H4 and H6 sites, respectively. The β-Proteobacteria, and γ-Proteobacteria subgroups, and the bacterial phyla Firmicutes, were the most abundant.

Interestingly, sites with higher salinity (H4 and H6) showed a greater diversity compared to the site with lower salinity (H3). Also, the lower similarities exhibited by obtained phylotypes to their closest relatives, may indicate the novelty of the sequences retrieved from this system. This work highlights that Salar de Huasco represent a potential reservoir of undescribed microorganisms for more detailed studies related with novel osmoadaptative mechanisms and biotechnological applications, a trend probably due to the unusual physicochemical conditions and geographical isolation of this wetland.

079A Microbial dynamics during the annual freeze in Bratina Island ponds Stephen Archer1, Craig Cary1, Ian McDonald*1, Karl Safi2, Ian Hawes3 1University of Waikato, New Zealand, 2National Institute of Atmospheric Research, New Zealand, 3University of Canterbury, New Zealand

The Bratina Island ponds located in the depressions of the McMurdo Ice Shelf, Antarctica go through an annual total freeze-thaw cycle resulting in significant and progressive changes which alter their physical, chemical and biological environments. A number of ponds were monitored from January to April in 2008, during this time light duration reduced to near darkness, temperatures within the ponds fell to below zero degrees Celcius, and conductivity increased due to the >80cm of downward ice growth. Molecular techniques were employed on these samples to investigate the bacterial community changes over this time. DNA fingerprinting information was coupled to a number of geochemical variables to identify drivers within the community and high throughput sequencing of selected samples has been employed to investigate specific community changes over this time period. 454 pyrosequencing of 4 samples created a dataset of 53,329 reads which was clustered into 1,815 OTUs at a distance of 0.05. Total read numbers indicate that the 20OTUs found in all samples represent over half of the sequences produced. Major shifts in community structure included the decrease in Cyanobacteria and increase in Euryarchaeota and unassigned groups with time. Over the entire time period there were no outstanding geochemical drivers of microbial diversity implying that the environmental drivers of the communities are different over time during the winter freeze up.

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080A Bacterial and archaeal community structure along a geochemical gradient in hydrothermally influenced sediments of Guaymas Basin, Gulf of California Philip A. Arevalo*1, Sean P. Sylva1, Caroline R. Toney1, Nadine Le Bris2, Jeffrey S. Seewald1, Stefan M. Sievert1 1Woods Hole Oceanographic Institution, USA, 2Benthic Ecogeochemistry Laboratory, Universite Pierre et Marie Curie, France

In many sedimentary environments, hydrocarbons can reach high concentrations and are potentially important substrates for microbial communities. One of the most spectacular places where large quantities of hydrocarbons are naturally introduced into the biosphere is the deep-sea hydrothermal vent site in Guaymas Basin, Gulf of California. Hydrothermal heating of immature sedimentary organic matter causes the active generation of liquid and gaseous aliphatic and aromatic products that are compositionally similar to crude oils in conventional petroleum producing basins. These petroleum-like products, which include gasoline-range aliphatic and aromatic hydrocarbons and residual polar asphaltic materials together with high amounts of NH4

+, are carried to the sediment surface by the ascending hydrothermal fluid. Thus, surface sediments contain large concentrations of C1-C10 hydrocarbons, and concentrations of C2-C7 hydrocarbons are about 10-10,000 times higher in geothermally warm Guaymas Basin sediments in comparison to the low concentrations (0.1-10 ppb per compound) of geothermally cold seafloor sediments. Four sediment cores were taken with HOV Alvin at the Cathedral Hill vent site along a transect from the vent into the surrounding sediment. Geochemical parameters were either determined in situ (sulfide, oxygen, pH) using voltametry or from porewater samples taken at specified depths down core. Sediment cores were sliced and nucleic acids extracted for subsequent 16S rRNA pyrotag amplicon sequencing to assess the diversity of bacteria and archaea and to understand how microbial community structure changes along the transect. Physical distance between cores on the transect may most strongly influence microbial community composition. Alternatively, temperature and associated geochemical parameters may also play an important role and thus microbial communities may be most similar on thermal isoclines along the transect. The results demonstrate that a diverse set of microbes populates this dynamic environment. Using multivariate techniques, we show that a sharp change in microbial community composition occurs at the site furthest from the vent. Among the three sites closest to the vent, however, temperature appears to play a larger role in influencing microbial community composition. For example, epsilonproteobacterial operational taxonomic units (OTUs) compose <5% of the community at the site farthest from the vent, but become much more abundant at the other three sites in the transect. Additionally, using phylogenetic analysis, we were able to identify two deltaproteobacterial OTUs closely related to 16S rRNA sequence from a cultivated sulfate-reducer able to degrade propane and butane anaerobically. These OTUs appear most frequently at the site closest to the vent itself. Among the Archaea, unclassified members of the Euryarchaeota dominate the cooler, shallower sediments of the transect, while Crenarchaeota dominate deeper, hotter sediment layers.

081A Spatiotemporal dynamics of bacterial community in the very As-rich creek waters of Carnoulès mine, France Volant Aurélie*1, Bruneel Odile1, Desoeuvre Angélique1, Casiot Corinne1, Bru Nöelle2, Delpoux Sophie1, Héry Marina1, Javerliat Fabien3, Fahy Anne3, Elbaz-Poulichet Françoise1, Duran Robert3, Bertin Phillipe4, Laugat Béatrice3 1Laboratoire HydroSciences Montpellier UMR 5569, France, 2UMR CNRS 5142, France, 3EEM, UMR 5254, France, 4GMGM, UMR 7156, France

Acid Mine Drainages are one of the most pernicious forms of pollution in the world, and are widely accepted as responsible for costly environmental and socio-economical impacts. They are generated when the wastes from the mining and processing of sulfide ores come into contact with oxygenated water. Such waters are often characterized by acidic pH and generally contain very high levels of iron and toxic chemical compounds. Although very hostile to life, some microorganisms are able to thrive in this extreme environment. Their roles in the oxidation reactions of sulfide minerals leading to the bioleaching are well known, as well as their roles in the bioremediation of these polluted waters. However, despite microorganisms make critical contributions to ecosystem function through their participation in biogeochemical cycles, we still have only a limited understanding of the factors that control the spatiotemporal structure and diversity of microbial communities. For example, although it is clear that microbial community composition is influenced by environmental variation the question of how diversity changes along environmental gradients remains generally unresolved. Elucidating how

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biodiversity is distributed and the mechanisms underlying those patterns is a central goal of ecology. Our study was conducted on the Carnoulès mine (France, Gard). This site provides an outstanding example of adaptation of life to extreme environments, acidic pH (2-4), very rich in metallic elements and especially As, (up to 10000 mg/l, one of the most important concentrations in the world). Moreover, its configuration is such that the contamination of the small Reigous Creek, which rises at the basis of mine tailings, is attenuated along the continuum. Indeed, nearly 95% of the arsenic in solution disappears between the source of Reigous and its confluence with the Amous River, 1.5 km downstream. In this study we used T-RFLP and pyrosequencing of 16S rRNA genes to investigate the spatiotemporal dynamics of bacterial community in relation to the physicochemical parameters. Five stations located along the gradient were sampled at six sampling period over approximately two years. This work showed a spatial gradient of physicochemical conditions and highlighted a significant shift in bacterial community structure. Indeed the attenuation of pollution seems to be related to an increase of the diversity of the bacterial community. As expected owing to the water chemistry, the sequences recovered are mainly related to bacteria involved in the geochemical cycle of Fe, S and As like Acidithiobacillus, Leptospirillum, Ferrimicrobium, or Thiomonas, typical of acid mine drainage. This deep sequencing of PCR amplicon libraries also allowed detection of low-abundance populations. Indeed, many sequences obtained are related to sequences from uncultured, newly described organisms or recently associated with acid mine drainage. It will be interesting to focus on these organisms and to investigate their potential role inside the ecosystem. Thus, this study help us to better understand the functioning of this ecosystem, to identify the populations present in this extreme environment, their seasonal dynamics and their effect on the redox reactions of metals and metalloids.

082A Molecular diversity and cold-adaptation in permafrost bacteria from Taylor Valley, Antarctica Corien Bakermans*1, Mark Skidmore2, Suzanne Douglas3 1Pennsylvania State University, Altoona College, United States, 2Montana State University, United States, 3NASA Jet Propulsion Laboratory, United States

The goal of this study is to understand diversity of organisms present in Antarctic permafrost and assess their potential to be active at low temperatures using only DNA-based techniques. Because frozen environments tend to preserve nucleic acids, the use of RNA studies to discriminate between metabolically active and total organisms may not be valid at low temperatures. Furthermore, low growth rates (expected due to low temperatures) result in lower amounts of RNA available for analysis. Therefore, alternate techniques are required to examine active microbial populations in cold environments. The organisms that are active in cold environments are most likely cold-adapted. This cold-adaptation should be evident in their macromolecules (that is amino acid modifications that result in more flexible proteins) and, in the case of proteins, is directly present in their DNA.

In this study we examined the diversity and cold-adaptation of bacteria within permafrost from Taylor Valley, Antarctic (mean annual temperature -18°C). A standard 16S rRNA gene clone library was constructed to assess total population diversity, while a clone library of the RNA polymerase beta-subunit (rpoB) gene was constructed to assess population diversity and cold-adaptation. RpoB is found in most bacteria and semi-universal primers exist (and were modified in this study). To demonstrate the potential for microorganisms to be viable and active in situ, plate counts and respiration assays were also conducted.

The 16S rRNA gene clone library was dominated by the phyla Acidobacteria (58% of clones) and Gemmatimonadetes (29% of clones). The rpoB clone library was similar to the 16S rRNA gene library with Acidobacteria dominating (84% of clones) and α-Proteobacteria, Actinobacteria and Chloroflexi sequences also present. Notably, Gemmatimonadetes were not represented in the rpoB library, but were detected by targeting large PCR products for cloning. Subsequently, five different protein properties typical of cold-adapted proteins were examined in rpoB OTUs and compared to its closest relatives. Acidobacteria OTUs were significantly more cold-adapted than their (primarily mesophilic) relatives while OTUs from the α-Proteobacteria, Actinobacteria, Verrucomicrobia, and Chloroflexi were not. OTUs from the Gemmatimonadetes, CFB and plastids (from the Chlorophyta) were significantly more cold-adapted than their close relatives in two of the five protein properties. Plate counts conducted at 4°C on a variety of media (Luria agar to M9 minimal salts agar) ranged from 9600±100 to 1700±200 cfu/g. Respiration of 14C-acetate was evident at temperatures from -5 to 22°C with the maximum rate of respiration occurring at 15°C.

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The plate count and respiration data suggest that a variety of cells present in TV permafrost remain viable and culturable. Furthermore, the nucleic acid studies suggest that it is the Acidobacteria, the dominant OTU of both clone libraries, and the Gemmatimonadetes, CFB and plastids that may be active at the low in situ temperatures of Taylor Valley permafrost.

078B Enrichment and identification of psychrotolerant iron- and sulfur-oxidizing Acidithiobacillus strain from Chilean Altiplano Sergio Barahona1, Cristina Dorador1, Francisco Remonsellez*2 1Universidad de Antofagasta, Chile, 2Universidad Católica del Norte, Chile

The use of microorganisms for the extraction of base metals from low-grade sulfide ores is currently a technology applied in some copper Chilean mines, and during the last few decades this technology has become increasingly important due to the depletion of high-grade ores and the existence of huge natural reserves of copper in the form of secondary copper sulfides. These microorganisms can grow in low pH environments; use inorganic materials, such as ferrous iron, reduced sulfur, and hydrogen, as electron donors; can tolerate high concentration of heavy metals; and vary widely in their temperature ranges.

Some natural ecosystems present in the Andes Mountains, such as the Chilean Altiplano, meet environmental conditions for the development of leaching microorganisms. In this work, we obtained enrichments cultures of iron- and sulfur-oxidizing microorganisms from an acid river in the Chilean Altiplano. Bacteria identification was performed using PCR products of 16S rRNA clone libraries, and the sequences analysis revealed the presence of a microorganism related to the recently described psychrotolerant Acidithiobacillus ferrivorans. We isolated the Acidithiobacillus strain, and studied their ability to grown using iron and sulfur as energy source, and also we determined its ability to grow at temperatures ranged between 4 and 30°C, confirming the psychrophilic characteristic previously described.

Interestingly, bacterial oxidation of sulfide minerals at low temperatures has been reported and several acidophilic iron-oxidizing strains have been isolated that are capable of growth at low temperatures, therefore results of this study highlight the importance of a novel bacterial strain able to mediating mineral oxidation in low-temperature environments.

083A Direct and crossed effects of temperature, pressure and salinity on metabolic activity of anaerobic strains Vanessa Barsotti1, Sébastien Dupraz1, Catherine Joulian*1, Fabienne Battaglia-Brunet1, Claire Sergeant2, Francis Garrido1 1BRGM, France, 2CENBG, France

It has been already noticed, by comparing several studies, that the possibilities to identify microbial activity in deep subsurface environments is not always in agreement with the known limitations for life concerning pressure, salinity and temperature. For instance, it has been noticed that it is very unlikely to find living microorganisms when temperature and salinity are higher than 70°C and 120 g/L respectively, which demonstrates a combined negative effect of salinity and temperature. In order to understand the different stress effects and the specificity of these parameters, eight selected microbial strains (bacteria and archaea), representative of the subsurface metabolism (methanogenesis, sulfate reduction, thiosulfate reduction, fermentation), have been tested on different temperatures (40, 55 and 70°C), pressures (1, 90 and 180 bar) and salinities (13, 50, 110, 180 and 260 g/L). Experiments were realized accordingly to a set of full factorial plans, and, for each test, the microbial activities were estimated by dosing the main expected metabolic products (CH4, H2S and acetic acid), the pH and the optical density. Results show variable response depending of the strains. Curiously, some species that were not supposed to be piezophile (that is surface species) have demonstrated a positive reactivity when the pressure was increased. On the other hand, fermenters and thiosulfate reducers were proven to be the most tolerant metabolisms to the overall range of exposition compared to other strains. Nevertheless, general trends have been also been identified: the combined increase of pressure and temperature as well as the combined increase of pressure and salinity did globally provide synergetic effect on the strains activities. This leads to ask whether it is possible that the symmetrical behavior of these stresses is due to the sensitivity of one common structural

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(membranes) or metabolic target (compatible solutes) that is implicated in the microbial mechanisms of resistance and/or adaptation.

084A Does the capacity to induce lightning in clouds increase the adaptive potential of Pseudomonas syringae? Laurine Blanchard*1, Sandrine Demanèche1, François Buret1, Cindy E. Morris2, Timothy M. Vogel1, Pascal Simonet1 1Ecole Centrale de Lyon - Université de Lyon, France, 2INRA, UR407 Pathologie Végétale, France

Among Pseudomonads, the P. syringae species complex contains phytopathogenic bacteria that have been isolated from many natural fresh water reservoirs around the world including clouds, suggesting their spread by the water cycle. In clouds, they can act as condensation nuclei inducing the formation of small droplets from water vapor and as heterogeneous ice nuclei catalyzing the transformation of water into ice. Under stormy conditions, ice particles are transported into clouds by fast airflow and their collisions and cleavages induce electrical charge separation. The resulting high electrical fields due to electrical charge accumulation inside clouds or between clouds and Earth induce the formation of ionized channels where several high-intensity pulses can be delivered corresponding to the visible part of the phenomenon: lightning. Injection of an electrical current in soil due to lightning discharge was demonstrated to induce environmental DNA entry into bacterial cells by a process called electro-transformation. The aim of this study combining physical modeling and in vitro experiments was to determine if ice nucleation activity and subsequent consequences on cloud processes including lightning could allow P. syringae to acquire new genes in situ. The physical modeling of lightning confirmed the presence of electric field pulses (few kV/cm) in clouds, associated with lightning currents, suggesting that electro-transformation could theoretically occur in the large volumes represented by clouds. In addition, P. syringae exhibits an unusual resistance level to lightning in simulated conditions including icy media in comparison to another model bacterium (Escherichia coli) and resists several repeated electric shocks and is efficiently transformed by extracellular DNA. We will discuss the potential involvement of this electro-transformation mechanism for explaining the adaptive potential of this ubiquitous microorganism considering its populations density in clouds and various electro-transformation parameters including bacterial pore stability over time that could potentially favor DNA uptake during rain fall.

085A Elevational patterns of bacterial diversity on glaciers Dylan Bodington*1, Takahiro Segawa2, Nozomu Takeuchi3, Yuichi Hongoh1, Shiro Kohshima4 1Tokyo Institute of Technology, Japan, 2National Institute of Polar Research, Japan, 3Chiba University, Japan, 4Kyoto University, Japan

Yala Glacier is a summer-accumulation type glacier in the Langtan region of the Nepali Himalaya. The surface of the glacier has numerous cryoconite holes, which are formed by the increased melting due to dark, dust-like material on the glacier surface. These cryoconite holes provide a relatively stable environment that supports a biotic community including cold-tolerant insects, copepods, algae and bacteria. Microscopic analysis of the algal community on the glacier has shown a change in community structure with altitude. In this study, we used molecular techniques to analyse the bacterial community of Yala Glacier in detail and compare it with the communities on Gulkana Glacier (Alaska) and Nef Glacier (Patagonia), in order to identify bacteria specific to the extremely cold, oligotrophic conditions of the glacier, and to determine the effect of altitude on bacterial community structure. Surface snow and cryoconite samples were collected from numerous sites on the glaciers ranging from the terminus to the peak. The 16S rRNA gene was amplified by PCR using a Bacteria-specific primer set, and the products were cloned and sequenced. Sequences were analysed phylogenetically and statistically using ARB and the QIIME pipeline. Yala Glacier was dominated by Betaproteobacteria, Cyanobacteria, Bacteroidetes and Alphaproteobacteria; whereas Gulkana and Nef were dominated by Bacteroidetes, Alphaproteobacteria, Betaproteobacteria and Acidobacteria. Dominant OTUs that constituted more than 1% of the total clone library of the glacier were chosen, to reduce species dispersed from the atmosphere which probably do not grow on the glacier. Many of the dominant OTUs showed more than 97% sequence similarity to sequences obtained from cold environments in public databases. Yala and Gulkana glaciers exhibited similar patterns of change in bacterial diversity with change in altitude. The frequency of dominant OTUs also varied with altitude, and showed a separation into three zones; upper, middle and lower, which correspond to the snow-covered accumulation area, snow- and ice-covered area around the snowline and ice-covered ablation

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area. The higher diversity in the middle zone could be explained by the intermediate disturbance hypothesis; relatively unstable conditions around the snowline may suppress competitive exclusion by the predominant species. The very low diversity in the upper zone could be due to repeated covering by fresh snow. Communities in the upper zones of the three glaciers showed greater similarity to each other than to the communities of the lower zones of the same glacier, while communities in the middle and lower zones were clustered by glacier, with Yala distinctly separate from Gulkana and Nef. The bacterial communities on the glaciers showed a significant change in diversity and species composition with altitude, with OTUs adapted to grow in cold conditions dominant.

086A Linking Mn(II)-oxidizing bacteria to natural attenuation at a former uranium mining site Tsing Bohu*1, Denise Akob1, Andrea Beyer2, Franziska Schäffner3, Matthias Haendel3, Carol Johnson4, Dirk Merten5, Georg Buechel6, Kai-Uwe Totsche6, Kirsten Kuesel1 1Friedrich Schiller university Jena/Institute of Ecology, Germany, 2Friedrich Schiller university Jena/ Institute of Microbiology, Germany, 3Friedrich Schiller university Jena/Institute of Earth Sciences, Germany, 4Virginia Tech/Department of Geosciences, USA, 5Schiller university Jena/Institute of Earth Sciences, Germany, 6Schiller university Jena/Institute of Geosciences, Germany

Natural attenuation of heavy metals is occurring in Mn oxide rich layers at pH ~5 in the subsurface of former uranium mining district Ronneburg, Germany. It is unknown how these Mn-rich layers are formed. As microorganisms readily oxidize Mn(II) at pH 7, we sought to determine if Mn(II)-oxidizing bacteria were present and could precipitate Mn oxides under in situ conditions. Therefore, in this study, we (1) characterized microbial communities in the moderately acidic (pH 5) Mn-rich layer; (2) tested pH and heavy metal tolerance of isolated Mn(II)-oxidizing bacteria; and (3) identified biogenic Mn oxides. Bacterial 16S rRNA gene cloning and isolation showed the dominance of β-Proteobacteria and Actinobacteria in the Mn-rich layer, including known Mn(II)-oxidizing bacteria. Cell abundance was low (8.5×105 cells g soil-1 based on quantitative PCR) and Mn(II)-oxidizing bacteria cultured at pH 5.5 and 7 represented 0.01% of the total bacterial community. Cultivation at pH 5.5 yielded two Mn(II)-oxidizing b-Proteobacteria isolates, AB_14 and TB-2, and at pH 7 we obtained 9 strains related to members of the Proteobacteria, Actinobacteria, and Bacteroidetes phyla. The closest relative of the pH 5.5 isolate TB-2 was Rhodoferax ferrireducens, which represented 16% of total bacterial clones in the Mn-rich layer. Our Mn(II)-oxidizing isolates tolerated a wide range of heavy metal concentrations, ranging from 1 to 100 fold higher than in situ porewater concentrations (85 µM nickel, 0.44 µM cadmium, 23 µM zinc, and 0.8 µM copper). The isolates growing at pH 5.5, AB_14 and TB-2, were sensitive to Cd and Ni, respectively, but could tolerate high concentrations of the other metals. The isolates grew across a wide pH range, from pH 5-8, with strains AB_14 and TB-2 growing in moderately acidic pH down to pH 5.0 and 5.5, respectively. Laser ablation inductively coupled mass spectrometry showed that isolates accumulated Mn and Fe in their biomass. Transmission electron microscopy analysis techniques including imaging, energy-dispersive X-ray spectroscopy and selected area electron diffraction showed variable morphology of precipitates confirmed the presence of manganese oxides. Isolate AB_14 was not surrounded with precipitates; whereas our Actinobacteria isolate AB_18 growing at pH 7 was encrusted with manganese oxides. Among these were highly crystalline Mn oxides of varying phase including groutite formed by AB_18 and possibly birnessite by AB_14. Preliminary Fourier transform infrared spectroscopy analysis indicated that all isolates formed precipitates with absorption bands characteristic for birnessite-like minerals. The ability of our isolates to grow at similar to in situ pH (5.0-6.0) and produce Mn oxides supports their environmental relevance. Since chemical oxidation is predicted to be slow at acidic pH, heavy metal tolerant Mn(II)-oxidizing bacteria may be involved in forming Mn oxides which are linked to natural attenuation in the former Ronneburg mining area.

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087A Identification and cultivation of thermophilic Planctomycetes Elizaveta Bonch-Osmolovskaya*1, Galina Slobodkina1, Margarita Miroshnichenko1, Olga Kovaleva1, Alexander Merkel2, Anna Perevalova1, Yana Nepomnyashchaya1, Alexander Slobodkin1, Nikolay Chernyh1, Andrey Mardanov3, Vitaly Kadnikov3, Esta van Heerden4, Olga Karnachuk5, Alexander Lebedinsky1 1Winogradsky Institute of Microbiology RAS, Russian Federation, 2Winogradsky Institute of Microbiology, Russian Federation, 3Centre , Russian Federation, 4University of the Free State, South Africa, 5Tomsk State University, Russian Federation

Bacteria of the phylum Planctomycetes are ubiquitously present in diverse ecosystems of the Earth where they play significant environmental role either degrading organic substrates or as anaerobic oxidizers of ammonium (“anammox bacteria”). All cultured Planctomycetes are mesophilic aerobic organotrophs, including Isosphaera pallida isolated from a hot spring and having its temperature optimum of growth at 40oC. However, there is molecular evidence of the presence of Planctomycetes in diverse thermal habitats including anaerobic ones: deep-sea and shallow-water hot vents, terrestrial hot springs, naturally composting sugar cane bagasse piles, deep subsurface environments.

Samples from thermal environments were obtained from terrestrial hot springs of Kamchatka (Uzon Caldera, Geyser Valley)and Kunsahir Island (Kurils), an oil well in Tomsk region (Siberia, Russia), and Beatrix gold mine (South Africa). For the detection of Planctomycetes in various thermal habitats and enrichment cultures we used PCR with various sets of 16S rDNA specific primers and consequent DGGE analysis of amplificate or pyrosequencing of the V3 region of the 16S rRNA genes. When studying the water of Parabel oil well (Tomsk region, Russia) we obtained a metagenome of microorganisms collected from the formation water. Enrichment cultures were obtained on anaerobically prepared medium with polysaccharides, sugars, or acetate as the energy sources; in some acetate-utilizing enrichments Fe(III) oxide was added as the electron acceptor.

In environmental samples and enrichment cultures, 16S rRNA gene sequences of Planctomycetes were detected and compared with the GenBank database. All the 11 sequences were found to be closely related to the environmental 16S rRNAgene clone OPB17 from Yellowstone National Park (96-100% similarity). Two isolates – R1-3 from Kunashir Island spring (100% similarity with OPB17) and BH1-P from Beatrix mine (96% similarity with OBP-17) were obtained in pure cultures. Both grow anaerobically on sugars and polysaccharides at 50-65oC and represent a novel genus in the phylum Planctomycetes.

These results show that thermophilic, phylogenetically close Planctomycetes are widely spread in terrestrial and subsurface thermal environments. Being anaerobic organotrophs, they participate in the degradation of organic matter in these habitats.

088A Restriction Endonuclease TypeII from Thermotolerant Soil Bacteria Isolated from Hot Springs in Northern Thailand Sakunnee Bovonsombut*1, Yingmanee Tragoolpau1, Panmuk Vacharapiyasophon1, Sittisin Bovonsombut2, Woottichai Nachaiwieng1, Fumio Kato3, Khozo Kanda3 1Chiang Mai University, Thailand, 2Maejo University, Thailand, 3Saga University, Japan

Restriction endonuclease is an enzyme that recognizes and cleaves a double strand DNA at a specific sequence. Restriction endonuclease type II is the most specific endonuclease enzyme that cleaves at the recognition sequence. In order to searching for novel restriction endonuclease typeII or more available isoschizomer, screening of restriction enzyme was carried out with 47 isolates of unidentified soil bacteria isolated from hot spring soils in Chiang Rai and Mae Hong Son provinces after the soils were enriched in half strength nutrient broth at 55°C for 9 days with 3 times subsequence to the same fresh medium. The endonuclease type II activity was found in nine strains that was classified in 2 groups based on their DNA digestion pattern in gel electrophoresis. To determine the recognition sequences of type II restriction endonuclease, the enzyme were partial purification performed based on column chromatography using phosphocellulose and Sephacryl-S 200 column, respectively. Digestion patterns of various DNAs, λDNA, ColE1, pBR322, φX174 and pUC19 with the individual enzymes isolated indicated that the first pattern from isolate TP011NA, is an isoshizomer of ClaI and the second pattern from the isolate KJ012, is an isoshizomer of NspV. Moreover both of enzymes, TP011NA and KJ012, showed activity from 37-75°C and had thermostability point at 60°C 20 minute.

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and 70°C 15 minute. respectively. The 16S rDNA analysis showed that the isolate TP011NA is a Bacillus sp. YMY1010 and isolate KJ012 is an Aneurinibacillus thermoaerophilus DSM 10154T with 99% similarity.

089A Diversity of microbial communities in coal mine drainage remediation systems treating elevated manganese Dominique Chaput*1, William Burgos2, Colleen Hansel3, Cara Santelli1 1Smithsonian Institution, USA, 2Pennsylvania State University, USA, 3Woods Hole Oceanographic Institution, USA

Water discharging from abandoned coal mines can contain extremely high manganese levels, and removing this metal is an ongoing challenge. Passive Mn remediation systems promote the growth of certain bacteria and fungi that catalyze the transformation of soluble Mn(II) to insoluble Mn(III/IV) minerals, but system performance is unpredictable. Mn-oxidizing organisms likely belong to complex communities that include algae, archaea, and bacteria and fungi that do not oxidize Mn. Community interactions could be modulating the efficiency of Mn oxidation in the treatment beds. Using a metagenomics approach, we aimed to determine the diversity and composition of microbial communities inhabiting several different Mn remediation systems, compared to surrounding uncontaminated soil, and to establish the abundance of known Mn-oxidizing microorganisms relative to the entire community.

Three Mn treatment beds in central Pennsylvania were selected, each treating metal-contaminated discharge from abandoned coal mines. Rock or sediment samples were collected near the influent, in the middle and near the effluent of each bed, as well as from surrounding uncontaminated soil. Amplicon pyrosequencing was carried out with primers targeting bacteria (small subunit rRNA), archaea (small subunit rRNA), fungi (internal transcribed spacer) and algae (large subunit rRNA). A variety of OTU- and phylogenetic tree-based analyses were performed to examine the composition and distribution of microorganisms inhabiting the treatment systems.

OTU-based analyses (rarefaction, Chao1, diversity indices) showed diversity levels in the treatment beds comparable to those in the surrounding soil, with no clear differences between or within beds. Clustering of samples by various similarity measures, followed by hypothesis testing, revealed no significant differences. These findings held for all four clades, regardless of the cutoff used for OTU binning. However, all samples (including those from the same bed) were significantly different in tree-based analyses (parsimony, Unifrac), pointing to high sample heterogeneity (as observed in soils). Proteobacteria dominated the bacterial profiles, followed by Acidobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria and Verrucomicrobia. Archaeal taxa were mainly unidentified, even at the phylum level. Ascomycota, Basidiomycota and Glomeromycota were dominant fungal phyla, and the algal profiles included high abundances of Bacillariophyta (diatoms), Phaeophyceae (brown algae), Xanthophyceae (yellow-green algae) and Chlorophyta (green algae). Sequences similar to Mn-oxidizing fungal and bacterial strains isolated from these same treatment beds accounted for a tiny proportion overall (<0.1% in most cases).

The high diversity of Mn treatment bed communities and their similarity to surrounding soil communities suggests that a wide range of taxa can tolerate high metal concentrations, perhaps through niche differentiation (e.g. by occupying pore spaces in the limestone) or through the detoxifying activity of Mn-oxidizers. The small proportion of known Mn-oxidizers is puzzling. Given the abundance of biogenic Mn oxides in these systems, either 1) the activity of these organisms is disproportionate to their relative abundance, or 2) Mn oxidation is carried out by far more taxa than have proven amenable to laboratory cultivation.

090A Extremely hypersaline isolates from microbial mats exhibit quorum sensing quenching and anti-microbial properties Sergey Dobretsov*, Raeid Abed, Marwan Al-Fori Sultan Qaboos University, Oman

In this study we tested the ability of hypersaline Archaean and bacterial isolates from microbial mats in Wadi Maqshan, Sultanate of Oman to exhibit anti-bacterial, anti-diatom, anti-algal and anti-quorum sensing activity. Microbes were isolated from the microbial mats and grown in modified growth

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medium (MGM) at 25% salinity. Pure Archaean and bacterial strains were identified based on their 16S RNA and morphological and physiological properties. In order to screen the bioactivity of these isolates, they were grown for 7 days in MGM with 25% NaCl at 35o C. The cells were separated from spent culture medium using centrifugation and separately extracted with hexane, dichloromethane and buthanol. Bioactivity of these fractions was investigated in laboratory experiments with nine human pathogens, the green microalga Dunaliella salina, the diatom Amphora coffeaeformis and quorum sensing (QS)reporter strains. QS quenching compounds were purified using high performance liquid chromatography (HPLC) and identified by comparison with existing C13 and H1 NMR and GC-MS data. Six isolates were obtained and they belonged to the genera Haloterrigena and Halomonas. All extracts exhibited growth of some human pathogens (Staphylococcus aureus, S. pyogenesis, and Bacillus subtilis). Only butanol extracts of spent culture media of one of the isolates (SK1) inhibited growth of the microalga D. salina; other extracts either did not affect the growth or induced it. In most of cases, extracts either induced or had no effect on the growth of the diatom A. coffeaeformis. Most of isolates inhibited QS of Chromobacterium violaceum CV017. Purification of dichloromethane extracts of one of the isolates (SK3) resulted in isolation of 4 different diketopiperazines (DKPs). Cyclo (L-Pro-L-Phe) and cyclo(L-Pro-L-iso-Leu) inhibited QS dependent production of violacein by C. violaceum CV017. Cyclo(L-Pro-L-Phe), cyclo(L-Pro-L-Leu),and cyclo(L-Pro-L-iso-Leu) reduced QS dependent luminescence of the reporter Escherichia coli pSB401 induced by 3-oxo-C6-HSL. None of isolated DKPs affected QS dependent luminescence of the reporter E. coli pSB1075 induced by 3oxo-C12-HSL. Our study suggested that extremely halophylic isolates produce anti-bacterial and QS quenching compounds, by which they possibly regulate behaviour and densities of other organisms in microbial mats.

091A Studying structure of methanotrophic communities from thermal springs of Uzon volcano caldera, Kamchatka Ekaterina Dvorianchikova*, Anna Kizilova, Irina Kravchenco, Valery Galchenco Winogradsky Institute of Microbiology, Russian Academy of Sciences, Russian Federation

Methane is the major greenhouse gas accumulating thermal radiation over 20 times more efficient than carbon dioxide, that points to its active impact on Earth’s climate. Studies, carried out in the last decade, revealed a new natural source of methane connected with flux of magmatic juvenile gases to the atmosphere, which are linked with breaks of crust and actively enter in the atmosphere in regions of geothermal activity.

Kamchatka peninsula is the unique area of modern volcanism manifestation. Kamchatka hosts more than 300 volcanoes in total. About 30 volcanoes are active and potentially active. Volcanoes which are extinguished long time ago are also of scientific interest, for example, Uzon volcano caldera, which appeared 40 000 years ago. On this area the great number of boiling mud springs, numerous mud pools with temperature from 45 to 98оС is observed. About 160 groups of springs with temperatures up to 98 оС in which various communities of thermophilic microorganisms develop are known now.

Recent studies showed in situ consumption of methane and methanotrophic activity in hydrothermal objects of various places (Italy, New Zealand, Kamchatka). However, knowledge on structure of methanotrophic communities of such ecosystems remains very limited.

The aim of this research consisted in studying structure of methanotrophic communities of thermal springs of Uzon volcano caldera (Kamchatka).

We collected silt and water samples from 36 thermal springs, located in the area of Uzon caldera. The springs differed in temperatures (37оС to 86.6оС) and рН (from 2.6 to 6.8).

Measurements showed existence of dissolved methane (from 5.19 to 71.58 μl/l) in all studied samples, therefore, they were analysed by real time PCR for presence of pmoA, a methane oxidation gene marker. Despite availability of methane, methanotrophs were found only in 8 springs. Samples in which methanotrophs were found, are divided into two groups – high-temperature neutral thermal springs (group 1) and low pH springs with moderate temperature values (group 2). Springs of each group were located closely to each other.

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FISH method allowed giving more differentiated analysis of methanotrophs in silt of springs. We found methanotrophs which belong only to Gammaproteobacteria, while the quantity of II type methanotrophs (Alphaproteobacteria) was below detection limit.

Analysis of translated amino acids sequences of PmoA showed that methanotrophic bacteria of Methylothermus genus, closely related to representatives of two valid species, are widespread in thermal springs of group 1. Other methanotrophs of these springs belong to other genera. In springs with low рН values (group 2) methanotrophic Gammaproteobacteria most closely related to Methylomonas and Methylobacter genera were found for the first time.

Results of our studies considerably expand existing ideas of variety of methanotrophs in extreme habitats, in particular, in hot springs located in Uzon volcano caldera, Kamchatka.

092A Metagenomic reconstruction of microbial communities from a CO2-driven geyser extends the environmental distribution of the Zetaproteobacteria Joanne Emerson*, Brian Thomas, Jillian Banfield University of California, Berkeley, USA

The recently discovered Zetaproteobacteria class has thus far only been detected in deep marine environments, including iron-rich, low-temperature hydrothermal vents and deep-sea sediments. Through metagenomics, we report near-complete genomes from microbial communities dominated by Zetaproteobacteria from a CO2-driven geyser. Our study site, Crystal Geyser (Green River, Utah, USA), is a cold (17°C), iron-rich geyser that erupts approximately 1-3 times per day, due to pressure from soluble and free-phase CO2 accumulation in an aquifer ~500 m below the surface. We collected 65 L of geyser water as it precipitated during an eruption in November 2009, and we sequentially filtered the water through 3.0 and 0.2 µm. DNA was extracted from the 0.2 µm filter, from which we generated 13 million 150 bp paired-end Illumina sequencing reads. We used the EMIRGE algorithm to reconstruct near full-length 16S rRNA genes from our metagenomic data and concluded that the geyser community is relatively low-diversity, containing approximately four organisms at 10% abundance or higher and 13 organisms at 0.1% abundance or higher. The best BLAST hits for these 16S rRNA gene sequences were to anaerobic and/or chemolithoautotrophic bacteria, including iron oxidizers, sulfur oxidizers, and sulfate and nitrate reducers. Metagenomic assembly resulted in significant genomic reconstruction of the dominant organisms, including 12 contigs > 100,000 bp with 24-42x coverage. Most large contigs had protein sequence similarity to Mariprofundus ferrooxydans, the sequenced representative of the iron-oxidizing Zetaproteobacteria, suggesting that we have reconstructed the first near-complete Zetaproteobacterial genome from a natural system. Interestingly, comparative genomic analyses suggest that, although many proteins are shared between the sequenced Mariprofundus ferrooxydans genome and contigs from Crystal Geyser, most are not syntenous, suggesting significant diversity within this clade. Our results extend the environmental distribution of the Zetaproteobacteria to include a CO2-driven, cold-water geyser and suggest that these organisms are likely to be abundant in the subsurface aquifer from which the geyser water originated. This study provides additional insight into the metabolic potential of the Zetaproteobacteria and places them in the ecological context of a natural, chemosynthetic ecosystem.

093A Microbial communities associated with alkaline hydrothermal systems of Prony Bay, New Caledonia Gael Erauso1, Anne Postec*1, Marianne Quémeneur1, Fatma Benaïssa1, Jérôme Hamelin2, Bénédicte Menez3, Emmanuelle Gérard3, Martine Gérard4, Bernard Pelletier5, Claude Payri5, Marie-Laure Fardeau1, Bernard Ollivier1 1Aix-Marseille University, Mediterranean Institute of Oceanography, 163, Av de Luminy, France, 2INRA, UR050, Laboratoire de Biotechnologie de l'Environnement, France, 3Institut de Physique du Globe Paris (IPGP), France, 4Institut de Minéralogie et Physique des Milieux Condensés (IMPMC), France, 5Institut de Recherche pour le Développement (IRD), UR 227, French Polynesia

A shallow submarine hydrothermal field comparable to that of Lost City (http://www.lostcity.washington.edu/) was discovered in Prony Bay in the south of New Caledonia (SW Pacific). Both Prony and Lost City are ultramafic hydrothermal systems driven by serpentinization reactions of water with mantle rocks resulting in warm, anoxic, highly alkaline fluids (up to pH 11) rich

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in calcium, dissolved hydrogen and methane but depleted in carbon oxides and metals. Thus they sharply contrast with the very hot, acidic metal rich fluids typical of sulfidic vents (“black smokers”) associated with magmatic heat. Like in Lost City, the Prony Hydrothermal Field (PHF) is characterized by large carbonate chimneys (up to 30 m in height) venting clear fluids, which temperature however do not exceed 40°C (while it can reach up to 90°C in LCHF). The main difference between the two sites is the geological setting: oceanic (800 m depth) on ultrabasic massif off the MAR for LCHF and a costal environment (0 to 50 m depth) on perodotite nappe in a tectonic active area for PHF.

Here, we report the first investigation on the microbial communities inhabiting the carbonate chimneys in PHF, focusing on the inner parts, most alkaline and warmest. For spatio-temporal analyses, samples were collected (2005, 2010 and 2011), from different sections of chimneys (top to bottom, outer to inner), located in seven sites (1 to 5 km distant) at varied depths (0 to 47 m) in the Prony bay. The structure and diversity of microbial communities and functional groups were compared by fingerprinting (SSCP/DGGE) and sequences analyses of clone libraries of both 16S rRNA and functional genes for methanogenesis (mcrA), methanotrophy (pmoA), and sulfate-reduction (dsrAB). We found that overall microbial patterns varied little inside a given chimney but changed in time and dramatically differs from one site to another, likely due to variation in hydrothermal activity and local geological settings.

In each site and chimney sample, the specific richness of bacteria was an order higher than of archaea, in agreement with previous studies on LCHF. Archaea were dominated by few phylotypes of Methanosarcinales, related to the ANME group 2 (responsible for anaerobic methane oxydation) Lost City Methanosarcinales (LCMS). Bacteria were mostly affiliated to Firmicutes, α-, β-, and δ-Proteobacteria. Uncultivated lineages(candidate divisions OP1 and OP7), Chloroflexi and deep branching phylotypes were also identified. Interestingly, many bacterial phylotypes were related to alkaliphiles (Desulfonatronum, Alkaliphilus) or, surprisingly, to thermophiles (Thermotagales, Thermus) of volcanic hydrothermal systems. The main metabolic groups were methylotrophs, sulphate-reducers, sulphide oxidizers for bacteria and methanogens or methanotrophs (ANME-related) for archaea indicating that microbial cycling of sulphur and methane must be the dominant biogeochemical processes in PHF as previously observed in LCHF. The overall functions occurring in this original microbial ecosystem resemble those reported for LCHF, meanwhile the microbial actors fulfilling these roles corresponds to unique phylotypes, endemic to PHF. These data provide an important initial microbiological description of a novel example of submarine alkaline hydrothermal ecosystem for meaningful comparison to the well-studied LCHF.

094A Geochemistry and microbiology in an acidic and high altitude (4.000m) salt flat in northern Chile Lorena Escudero1, Guillermo Chong2, Juan José Pueyo3, Cecilia Demergasso*1 1Centro de Investigación Científica y Tecnológica para la Minería (CICITEM) and Centro de Biotecnología-Universidad Católica del Norte, Chile, 2Departamento de Ciencias Geológicas-Universidad Católica del Norte, Chile, 3Departamento de Geoquímica-Universidad de Barcelona, Spain

In the arid Central Andean high plateau (Bolivia, Chile and Argentina, between 19° and 27° S latitude) there are more than one hundred endorheic basins with salt lakes and salt flats. Of these basins only the salt flats of Gorbea and Ignorado display strongly acidic brines (pH between 4 and 2). The cause of this local acidity has been attributed to the superposition of two factors: a) the significant volcanic native sulphur occurrences around their catchments, and the release of sulfuric acid by oxidation, and b) the strong hydrothermal alteration of the country rocks that lowered their buffering capacity.

The Salar de Gorbea (25 ° 24 'S, 68 ° 40' W) has an approximate area of 30 km2 and a catchment of around 320 km2. Country rocks in the W and S parts of the catchment show strong argillic alteration with high amounts of alunite, opal, gypsum, hematite, kaolinite, and minor amounts of jarosite, birnessite and other more soluble salts. Brines into the salt flat are of the Cl-SO4-Na(-Mg) type containing high concentrations of aluminium and boron, lower concentrations of manganese, lithium and iron, and still lower arsenic levels. Median values of Cl/SO4, Al/Fe and Mn/Fe are 1.22, 49 and 5, and are different from the Australian hydrochemical equivalents described in the literature (5, 5 and 0.05, respectively). Surface sediments are mostly composed by an irregular layer of primary selenitic gypsum, up to 2 m thick, that covered in the past the entire surface of the salt flat. Now is largely

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destroyed by dissolution, resulting in ephemeral ponds surrounded by efflorescences formed by halite, magnesium sulfate (mainly hexahydrite), sodium sulfate (thenardite) and magnesium chloride (carnallite). Lower amounts of aluminium and magnesium chloride-sulfates (kainite, magnesioaubertite) and very low amounts of Fe-bearing chloride-sulfates were also evidenced by XRD and SEM-EDS.

Jarosite has been found beneath the gypsum layer, and more soluble Al and Fe-bearing salts occur also as efflorescences around the ponds. By contrast, native sulphur and alunite are only present in the outer part of the catchment. The occurrence of jarosite and yellow efflorescences could be microbial mediated. Signatures of jarosite as well as magnesium salts similar to the described here were recognized by Compact reconnaissance Imaging Spectrometer for Mars (CRISM) in Noctis Labyrinthus.

The microbiome was investigated by direct count and culturing dependent (enrichment, isolation, oxidation rate determination) and independent techniques (DGGE, genome sequencing). The microbial community in brines and sediments (105 and 106 cells/mL respectively) were dominated by microorganisms close related to known acidophilic (Acidithiobacillus, Alicyclobacillus) plus others barely found in acidic environments (Pantoea, Mycobacterium, Geobacillus, Rhodanobacter). The lower salinity the higher microbial diversity was observed. Sulphur and iron oxidation activity were detected depending also on the salinity of the samples. Pure and mixed cultures have been obtained as a halotolerant strain of A. thiooxidans. Geochemical, mineralogical and microbial studies are going on to model the participation of microbial activity in the production of the local acidity.

095A From microbial communities to nanocrystals: a multi-scale approach towards understanding interactions among bacteria, iron minerals, and metal contaminants Maria Fabisch*1, Gina Freyer1, Carol Johnson2, Michael Hochella, Jr.2, Kirsten Küsel1 1Friedrich Schiller University Jena, Institute of Ecology, Aquatic Geomicrobiology Group, Germany, 2Virginia Tech, Department of Geosciences, Center for NanoBioEarth, USA

In the former uranium mining area Ronneburg, Germany, groundwater effluent with high ferrous iron and metal(loid) concentrations at pH 6.0 flows along a grassland into a small creek, forming iron-rich terraces at the creek bank. Iron oxyhydroxides, formed by iron oxidizing bacteria or chemical oxidation, may serve as important biogeochemical interfaces for adsorption or co-precipitation of heavy metals. We aimed to i) investigate the development of bacterial community structure with special focus on iron oxidizing and reducing bacteria, and ii) characterize colloidal and sedimented iron minerals and determine their heavy metal contents. Free-living and particle-associated organisms from water and fresh sediments were used for 16S rRNA gene cloning and sequencing. We found that members of the Proteobacteria (mainly Beta- and Deltaproteobacteria) dominated bacterial communities. Strikingly, high fractions of clones had ≥97% sequence similarity to reported iron oxidizing (32%) or reducing bacteria (5%). The neutrophilic, microaerobic iron oxidizer Gallionella sp. ES-2 was the most dominant iron organism (30%), especially in groundwater effluent (54%) and sediment of the creek site adjacent to the terraces (75%). The suboxic effluent harbored also Geobacter-related species, whereas clones related to Sideroxydans and Thiomonas sp. were detected more in downstream sediments. Quantitative PCR will allow us to follow the abundances of iron oxidizers and reducers over time, such as Gallionella sp., Ferrovum myxofaciens, or Geobacter sp. Analytical transmission electron microscopy of the effluent revealed the presence of 10-20 nm thin Zn-bearing sulfate green rust, a mixed Fe(II)/(III) mineral. In the water flowing over the terraces amorphous iron phases and nanocrystalline akaganeite and ferrihydrite were found. X-ray diffraction analysis of terrace sediments indicated the presence of goethite, and spheroidal particles of 100-1000 nm size had grown goethite nanoneedles. ICP-MS analyses of filtered water fractions showed that Al, Cr, Cu, Pb, and U were associated with particles (fraction > 0.45 µm). The metals of most concern, Ni and Zn, stayed in the dissolved/colloidal fraction, despite their potential for sorption to or co-precipitation with iron oxyhydroxides at pH 6. However, sediments and sediment porewater were enriched in heavy metals, for example Cd, Ni, or Zn. As iron oxidizing bacteria, especially Gallionella sp., represented such high fractions of the total bacterial communities, they appear to contribute to the formation of iron oxyhydroxides in the sediment influencing the later uptake of metal(loid) contaminants.

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095B Bacterial and archaeal diversity in permafrost soil from Kunlun Mountains Pass, Tibet Plateau of China Huyuan Feng*1, Weigang Hu1, Qi Zhang1, Jing Mu1, Gang Cheng1, Tian Tian1, Dingyao Li1, Fujun Niu2, Qingbai Wu2, Lizhe An1 1School of Life Sciences, Lanzhou University, China, 2State Key Laboratory of Frozen Soil Engineering, Chinese Academy of Sciences, China

Culture-dependent and culture-independent methods (Bacteria and Archaea 16S rRNA gene clone library analyses) were used to investigated the microbial diversity of three soil samples (KL1, KL3 and KL48) in a permafrost core from Kunlun Mountains Pass, Tibet Plateau of China. The 35 Bacteria isolates were phylogenetically related to the phyla Firmicutes, Actinobacteria, Proteobacteria and Cytophaga – Flavobacteria – Bacteroides (CFB) with Actinobacteria being the most abundant; all the isolates were psychrotolerant, the overwhelming majority of the isolates (27) were halotolerant. The three bacterial libraries containing 410 clones were composed of 72 phylotypes. Proteobacteria, Actinobacteria and Firmicutes were common to the three libraries and were the most predominant phyla (>10% of the clones). Besides,KL1 and KL3 shared the phyla Bacteroidetes and candidate phylum OP10. Phyla Planctomycetes, Acidobacteria, Gemmatimonadetes, Verrucomicrobia and Chloroflexi were present only in KL1. The three archaeal libraries containing 263 clones were composed of 14 phylotypes and contained sequences related to Thaumarchaeota, Euryarchaeota and Crenarchaeota. Thaumarchaeota was the most predominant phyla (>90% of the clones) in the three libraries. The majority of sequences in the Archaea libraries were related to ammonia-oxidizing archaeon (AOA). The results of principal component analysis (PCA) based on the 16S rRNA gene clone library data, percentages of phyla and biogeochemical data indicated that the samples from permafrost table (KL3) and permafrost (KL48) were different from the sample from active layer (KL1) and PH affected the observed heterogeneity in the percentages of phyla and the diversity of microbial communities in the soil samples. These reseaches on characterization of the microbial diversity establish the base to investigate the distribution and function of the microbial community in the permafrost of the Tibetan Plateau.

096A Survival of dry periods by iron depositing bacteria in biofilms attached to mars regolith simulants Nina Feyh*1, Bertram Schmidt1, Jean-Pierre de Vera2, Ulrich Szewzyk1 1Technische Universität Berlin/Institute of Environmental Technology, Environmental Microbiology, Germany, 2German Aerospace Center (DLR), Institute of Planetary Research, Germany

In surface water habitats with high Fe(II) concentrations, neutrophilic iron depositing bacteria frequently form large biofilms in which insoluble Fe(III)-oxyhydroxides are embedded. Iron bacteria can be found as well on temporarily dry sites. The extracellular polymeric substances of biofilms and the covering anorganic Fe(III)-compounds may be, besides other factors, an adaption to frequent desiccation and shading against UV radiation. In this work, iron depositing isolates are studied as model organisms for the feasibility of life in the martian subsurface. The influence of iron incrustation, biofilm formation and association to Mars regolith simulating particles on the survival of iron bacteria under desiccation stress is investigated.

Bacterial strains BS1, BS2, FL1, FL6 and B406 (genera Pseudomonas, Hyphomonas, Tetrasphaera, Pedomicrobium and Leptothrix) were isolated from different sampling sites (Tierra del Fuego/Argentina; Unteres Odertal, Bad Salzhausen/Germany). All strains show the ability to deposit ferrous iron under laboratory conditions.

Biofilms were grown in media supplemented with Fe(II) on pelleted Mars analog mineral mixtures (phyllosilicatic and sulfatic Mars regolith simulants). Survival of cells after desiccation was determined by plating onto agar medium and fluorescence in situ hybridization. Analyzes of multispecies biofilms showed a survival of cells after desiccation and dry storage for 1 month at room temperature and further 9 months at -20°C in a desiccated biofilm, detected both by fluorescence in situ hybridization and regrowth on agar plates. For biofilms grown on phyllosilicatic Mars regolith simulant 53,7% (sulfatic 81,2%) of total cell counts were active according to hybridization after recultivation and 15,8% (sulfatic: 0,5%) formed colonies on agar medium.

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Single strains as well as mixed cultures are currently examined in desiccation experiments considering iron deposition and mineral association as potentially influential parameters. The investigated iron depositing strains showed the ability to survive dry periods, which makes them interesting organisms for research on life under extreme conditions. The fluorescence in situ hybridization method proved useful to detect non culturable active cells after desiccation in biofilms.

097A Diversity of Aerobic Endospore-forming Bacteria in Geothermal Sites in Greece Sevasti Filippidou*, Nicole Jeanneret, Ludovic Roussel-Delif, Tina Wunderlin, Thomas Junier, Pilar Junier University of Neuchatel, Switzerland

The Greek territory is well recognized for its geothermal activity. Due to seismic and volcanic activity, natural hot springs occur frequently, distributed throughout the continental and island territory. Such environments constitute a diverse assemblage of microbial ecosystems. Temperature variations, from 15°C to 80°C, pH range from 3 to 9 and mineral composition at diverse natural sources have been described. Our aim was to study the distribution of aerobic endospore - forming bacteria at these sites, for which no data was available before. Sampling was conducted in12 natural hot springs in Northern Greece and on the volcanic island of Milos (south-west). Sampling sites were particularly diverse ranging from sites with luxurious vegetation and rivers to caves, inactive volcanoes and underwater geothermal sources. Moreover, human activity was observed in some of these sites, as they are used for recreational thermal baths. In total, 52 sediment, sand, soil and water samples were collected, of which 16 were retrieved from marine environments (underwater hot springs).

In order to study the microbial diversity and distribution by site, the methodological approaches were: Enrichment and isolation of bacteria on marine and nutrient agar; microscopic observation; DNA extraction; PCR amplification of 16S rRNA gene and a molecular marker for endospore-formation(the spo0A gene); sequencing and phylogeny.

In total, 80 strains have been isolated, from which 60 were identified as endospore-forming bacteria. In all 60 isolates, the spo0A gene was amplified and its sequence confirmed. These strains belong to Bacillus, Geobacillus, Anoxybacillus, Aneurinibacillus, Lysinibacillus, genera. DNA has been extracted directly from volcanic fumaroles and sediments, but no cultured strains could be obtained from these sites so far.

Phylogeny revealed the clustering of strains from distant sites, showing a temperature-based distribution pattern. Isolates from a high temperature drilling (85°C, Zefyria, Milos) clustered together with strains from high altitude natural springs (Thermia, Drama), where the only common characteristic was high temperature (above 60°C). Moreover, 10 Geobacilli strains, from distant natural sources with diverse environmental characteristics and temperature range between 35-40°C, also clustered together. Strains isolated from a marine environment (high salinity cave, Agia Paraskevi, Chalkidiki) cluster with an isolate from a thick mud lake (Pikrolimni, Kilkis). Temperature on both sites was between 25 and 35°C.

Human activity does not seem to be a contributing factor concerning distribution.

Overall, additional information from other molecular techniques, such as DGGE and full genome sequencing of our isolates, could provide additional information to understand the factors determining the biogeographical distribution of endospore-formers.

098A Bioprospecting for proteases from bacteria isolated from soil of St George Island, Antarctic Anderson Fragoso dos Santos*, Anderson Fragoso dos Santos, Daniela Costa Pinto, Hugo Emiliano Jesus, Angelo Samir, André Santos, Raquel Peixoto, Alexandre Rosado, Marta Branquinha Federal University of Rio de Janeiro, Brazil

The biomes on the Antarctic continent are subject to extreme temperature, humidity, UV irradiation, nutrient and pH conditions. Moreover, some of these parameters can change dramatically in short

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periods of time. Such extreme conditions, mainly of temperature, can be regarded to present strong selective forces shaping the microbial community make-up and diversity in the environment. These several distinct habitats have served as a potential region for identifying novel psychrophilic bacteria with biotechnological potential. Psychrophilic enzymes are characterised by high efficiency under low temperatures, high degree of thermolability as well as increased structural flexibility for better access to the substrate. Under low temperatures, protein flexibility plays a key role in adapting to cold. In spite of their undeniable potential, psychrophiles and their products are biotechnologically underused. However, developments in this area, mainly in psychrophilic enzymes, point to a rapid growth in this field. In this context, four of the 25 psychrophilic bacteria isolated from St George Island soil close to the Brazilian research station, showed high activity on qualitative tests (petri dishes containing protein substrates-casein and gelatin). These bacteria were designated 24, 38, 62 and 64. Comparative and philogenetic analysis of 16S RNA gene sequences revealed that those strains were Pseudomonas Antarctica, Psychrobacter fozii, Sporosarcina aquimarina and Algoriphagus antarticus respectively. The extracellular extracts of the culture supernatants from all isolates displayed proteolytic activities which were detected by (SDS-PAGE) sodium dodecyl sulfate polyacrylamidegel electrophoresis. P. antarctica showed two peptidases with molecular mass about 130 and 59 KDa. P. fozii and S. aquimarina showed one peptidase with 50 and 55 KDa respectively. All those peptidades demonstrated optimal activity at pH 7.0 at 22°C. A. antarticus also showed just one peptidase, however, its molecular mass was about 90 KDa and optimal activity at pH 9.0 at 37°C. Under these optimal activity conditions, the peptidases from all isolates were inhibited by phenanthroline and EDTA, two metallopeptidase inhibitors. Despite the relevance of the psychrophilic protease studies, this is the first account of proteinases obtained from these species. These results contribute to enhance the basic knowledge of proteases originating from the Antarctic environment as well as to help prospective future biotechnological applications of those two enzymes in particular.

098B Metabolic potential of halotolerant spore-forming gram-positive bacteria from Restinga, Rio de Janeiro, Brazil Anderson Fragoso dos Santos*, Roberta Valle, Vanessa Marques Alvarez, Lucy Seldin, André Santos Federal University of Rio de Janeiro, Brazil

Halophilic and/or halotolerant spore-forming gram-positive bacteria are particularly important because they can grow optimally in a wide range of salinity due to haloadaptation mechanisms which enable them to grow and survive in harsh environments. This group of bacteria is widely distributed in different saline habitats, such as hypersaline lakes, saltern ponds, Dead Sea water, the marine solar saltern of the Yellow Sea, surface saline soils, fermented seafood, desert iguanas, and mural paintings, causing biodeterioration.

Spore-forming gram-positive bacteria are especially useful in industrial processes due to their capability to produce secondary metabolites with diverse biological activities. Thus, searching for novel spore-forming bacteria is essential for the discovery of natural product-based drugs and/or enzymes.

The site chosen to search for microorganisms with potential enzymatic activity was Restinga da Jurubatiba - an environmental protected area in northern Rio de Janeiro State - which is considered to be the best preserved restinga area in the country for its huge intact biodiversity. Gram positive, halotolerant and spore-forming bacteria were isolated from the restinga soil, which generated 38 isolates of Halobacillus, Virgibacillus and Oceanobacillus genus. Due to the relevance of enzymes, the advantages of the ones obtained from microorganisms and the fact that proteases are among the three largest enzyme groups as well as account for 60% of the international enzyme trade, the goal of this project was to select among the 38 isolates the ones that displayed proteolytic activity. To do so, qualitative and quantitative tests (petri dish and SDS-PAGE containing protein substrates and chemical dosage of proteolytic activity) were carried out to screen proteolytic activity. Around 30% of the isolates displayed proteolytic activity on dish. The M9 isolate, Halobacillus blutaparonensis, displayed higher enzyme index and was selected for further proteolytic characterisation tests. Studies of H. blutaparonensis synectic cell growth showed growth in culture medium supplemented with different NaCl concentrations (0.5-10%), with optimal cell production at 2.5% NaCl. Three peptidases secreted with approximately 45, 35 and 28 kDa molecular masses were identified in this new bacterial species, through the use of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) containing gelatin substrate embedded in gel. These peptidases displayed the following biochemical properties: (i) wide pH spectrum activities (2.0-9.0 , highest hydrolytic activities in neutral pH), (ii)

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activities in different salt concentrations (0.5-20% NaCl), (iii) inhibition of 45 and 28 kDa peptidases by PMSF (a serine peptidase inhibitor) and 35 kDa peptidases by 1.10-phenantroline (a metallopeptidase inhibitor ), (iv) different protein substrate degradation (gelatin, casein, albumin), (v) the major peptidase (45 kDa) was the only one to degrade hemoglobin, its proteolytic activity was totally inhibited by ions Cu2+, Zn2+ and Fe2+ and partially inhibited by ions Co2+ in 10 mM concentration. The major 45 kDa serine peptidase also showed moderate thermostability and optimal stability to a wide pH range (5 - 9).How these microorganisms adapt to salinity variations and the knowledge of how these enzymes withstand such a variety of environments are key to many areas of biotechnology.

099A Induction of dormancy in ice wedge isolates and characterization of dormant cells Kentaro Funo*1, Indun Dewi Puspita1, Moe Uehara1, Wataru Kitagawa2, Michiko Tanaka1, Youichi Kamagata2 1Hokkaido university, Japan, 2Hokkaido university and National Insitute of Advanced Industrial Science and Technology, Japan

For a long time, studies of microbial ecology have revealed diversity and function of bacteria in natural environment, yet it remains unclear how they survive in the environment. An extreme environment like permafrost is not favorable for growth and division of bacteria because of the sub-zero temperature, low water activity, limited nutrient and limited oxygen concentration. However, A 25,000 year-old permafrost ice wedge was found to contain 106 CFU/ml viable cells and most of them were non-spore forming bacteria. We hypothesized that non-spore forming bacteria were able to survive in this extreme environment by entering a non-dividing state (viable but non-culturable state) to cope with environmental stress.

In a previous study, we found that a permafrost ice wedge isolate, Tomitella biformata AHU1821T, was able to enter a non-dividing state under oxygen-limited minimum medium conditions in a short period and to resuscitate by its own resuscitating promoting factor. To determine if this is a common survival strategy, in this study, we investigated the ability of other ice wedge bacterial isolates to enter a non-dividing state under different stress conditions.

Permafrost ice wedge isolates Arthrobacter sp. AHU 1770 and Glaciibacter superstes AHU 1791T were tested and Escherichia coli ATCC12435 was used as a positive control which is capable of entering a non-dividing state. Optimal growth conditions for Arthrobacter sp. and G. superstes were determined and used to grow cells prior to testing stress conditions. Then cells were incubated (up to 120 days) under oxygen limitation in modified minimum medium or shifting temperature to test stress conditions inducing cells into a non-dividing state. Throughout the incubation period, we calculated the number and proportion of non-dividing cells by comparing the total number of live cells from direct microscopic counts (Live/Dead staining) to dividing cells based on colony forming units (CFU).

All bacterial isolates could be induced into a non-dividing state in a short period. After oxygen limitation in modified minimum medium for 30 days, 99.9% of the Arthrobacter sp. cells were in a non-dividing state. The G. superstes cells went into a non-dividing state under two stress conditions, after oxygen limitation in modified minimum medium for 40 days and after 10 days incubation at an elevated temperature (30℃). The control, E. coli was incubated into a non-dividing state after 14 days of oxygen limitation in M9 and also after 30 days in M9.

From these results, Oxygen limitation in modified minimum medium and an elevated temperature appears to be a stress factor that induced the non-dividing state of ice wedge bacterial isolates.

100A Microbial diversity and enzyme characteristics of the unique ikaite columns; a permanently cold and alkaline environment in SW Greenland Mikkel Andreas Glaring*1, Jan Kjølhede Vester1, Jeanette Eva Lylloff1, Waleed Abu Al-Soud2, Søren Johannes Sørensen2, Peter Stougaard1 1University of Copenhagen, Dept. of Plant and Environmental Sciences, Denmark, 2University of Copenhagen, Dept. of Biology, Denmark

The ikaite columns in the Ikka Fjord, SW Greenland, constitute a permanently cold (4-6°C), alkaline (pH 10.4), and low salinity (0.9%) environment. The columns grow from the bottom of the shallow inner fjord and are composed of the mineral ikaite, an unusual metastable hexahydrate of calcium-

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carbonate. The columns are formed where alkaline, carbonate-rich groundwater seeping up from the fjord bottom meets the cold, calcium-rich marine waters of the Ikka Fjord.

Investigations of the ikaite columns have revealed a microbial community adapted to this extreme ecological niche. We have carried out a comprehensive investigation of the microbial diversity in the ikaite columns and the surrounding fjord by pyrosequencing of the V3 and V4 hypervariable regions of 16S rDNA (pyrotag sequencing). Analysis of the more than 500,000 high-quality tags generated have revealed a diverse environment with significant inter- and intra-column heterogeneity dominated by Alpha- and Gammaproteobacteria, Bacteroidetes, and Firmicutes. Several of the most common species found in the ikaite columns are related to alkaliphilic or psychrophilic species isolated from similar environments, such as alkaline soda lakes.

Analyses of a collection of cultivable isolates show a clear growth-preference for the cold and alkaline conditions characteristic of the ikaite columns. Many of the isolates also produce extracellular enzymes active at low temperatures and high pH making the ikaite columns a promising source of future industrial enzymes.

101A How do indigenious thermophilic microorganisms affect the permeability of potential CO2 storage sites? Claudia Gniese*1, Carsten Freese2, Nils Hoth3, Andrea Kassahun4, Martin Mühling5, Michael Schlömann5 1TU Bergakademie Freiberg / Institute of Biological Sciences, Germany, 2TU Bergakademie Freiberg / Institut f. Bohrtechnik und Fluidbergbau, Germany, 3TU Bergakademie Freiberg / Institut f. Bergbau und Spezialtiefbau, Germany, 4Dresdner Grundwasserforschungszentrum e. V., Germany, 5TU Bergakademie Freiberg / Institut f. Biowissenschaften, Germany

The controversial discussion about the carbon capture and storage (CCS) technology strongly advised politicians and researchers on great demands on safety measures and information in Germany. Hence, researchers are asked to investigate safety and integrity of potential CO2 storage formations such as depleted gas and oil fields.

In the BMBF-funded project CO2BioPerm, we address the influence of biogeochemical CO2 transformation processes on the long-term permeability behaviour of reservoir and cap rocks as well as of wellbore cements. Therefore, biogeochemical and geochemical flow-through experiments were examined simultaneously.

The experimental setup consisted of two identical drilling cores of Postaer sandstone (10cmx10cm, pores ~100µm, pore volume 140ml). Each core was lagged with a rubber coat and fixed vertically in a temperature-controlled retainer (≤70°C). The biogeochemical system was once inoculated with a pure culture of a Petrotoga isolate (inoculum: 2.8*106 cells/ml). The isolate originates from formation fluid of a wellbore of the natural gas field Schneeren (Lower Saxony, Germany; Gaz de France SUEZ).

The growth medium DSMZ 718 is specific for Petrotoga species and was pumped every two to four days through stainless steel pipelines from the bottom of each core to its top (10ml/min). The liquid medium was prepared aseptically under anaerobic conditions.

Liquid samples were collected at the outlet of the biogeochemical system to detect any variations in the microbial composition and at the outlet of the geochemical system to monitor the absence of microorganisms. Molecular-genetic and microbial analyses were performed using T-RFLP (terminal restriction fragment length polymorphism) and fluorescence microscopy (CARD-FISH, catalyzed reporter deposition fluorescence in situ hybridization). The permeability of the Postaer sandstone was monitored during every flow through.

Results showed no microbial contamination in the geochemical experiment and the permeability was 7*10-15m2.

In the biogeochemical experiment, a proportion of the Petrotoga isolate remained within the core as there were toga-like cells countable microscopically in the outflow even 34 days after inoculation. Cell

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numbers at the outflow ranged between 1.4*105 and 6.9*105 cells/ml. All detected cells were proven to be Petrotoga species using T-RFLP analyses.

The permeability behaviour of the Postaer sandstone varied between 4*10-15 and 6*10-15m2 in the biogeochemical system showing a similar level as in the geochemical system. Hence, no trend of the permeability towards higher or lower values was visible until now. This might change in the ongoing experiment or when using CO2 to simulate CO2 storage.

Using these preliminary results, we were able to overcome some technical obstacles and are now able to investigate the influence of CO2 transformation processes on the permeability of reservoir and cap rocks.

In addition, DNA of the Petrotoga isolate is presently submitted to genome sequencing. Using the genome sequence, it will be possible to obtain information on the effect of CO2 on gene expression in Petrotoga.

102A Microbial Activity and Diversity through a cold, hyper-arid desert permafrost core in University Valley, Antarctica Jacqueline Goordial*1, Alfonso Davila2, Margarita Marinova2, Dale Andersen3, Charles Greer4, Wayne Pollard1, Chris McKay2, Lyle Whyte1 1McGill University, Canada, 2NASA Ames Research Center, USA, 3SETI Institute, USA, 4Biotechnological Research Institute, National Research Council of Canada, Montreal, QC, Canada

The McMurdo Dry Valleys (MDVs) in Antarctica are the largest ice-free region of the Antarctic, and are characterized as the oldest, coldest and most arid environment on Earth. The open soils of the inland MDVs, to date, seem to be largely inhospitable to life. Surface soils experience wide temperature fluctuations, cold temperatures, extreme aridity, oligotrophic conditions, high incidence radiation and physical disturbance. The polar desserts located in the higher elevation mountains of the MDVs (the Upper Dry Valleys) are the only place on Earth where there is the presence of permanently frozen ice-cemented ground (permafrost) overlain with a layer of dry, ice-free permafrost soil. University Valley (UV) is an Upper Dry Valley, located at an elevation of 1650 m, with summer air temperature maxima never reaching above 0°C. At this site, ice in the ice-table exchanges with the atmosphere via water vapour diffusion rather than liquid water, thus this is a site of extremely low water activity. The potential for UV to support life at subzero temperatures is being investigated through a combination of culture dependent and culture independent approaches. A microbial community that is largely recalcitrant to culturing is being found in the MDV soil, as only 6 microorganisms have been isolated to date, two of which (Sphingomonas sp, Rhodococcus sp.) are capable of subzero growth down to -10°C, and are halotolerant. Microscopic cell counts have found low biomass across all samples, on the order of 104 cells/g. Radiorespiration assays using 14C-labelled acetate were carried out on environmental samples to measure microbial activity at 5°C and subzero temperatures. Activity assays are revealing an aerobic heterotrophic community that has none, or significantly lower activity when compared to those measured from maritime influenced Antarctic, and Canadian High Arctic permafrost samples. Next generation pyrosequencing was carried out throughout a permafrost core across all three domains of life. Sequencing analysis has revealed surprisingly high microbial diversity in subsurface permafrost samples, with the majority of bacterial families not being represented in more than one sample indicating high heterogeneity in microbial composition throughout the permafrost profile. The majority of bacteria in subsurface permafrost samples were found to compose a small proportion (1-2%) of total bacterial OTUs present, with the exception of a sample at the ice-table and dry surface soil interface which was enriched for the methylotroph family Methylophilaceae (26%). While the subsurface permafrost samples showed remarkably high diversity, the desiccating surface soil above the permafrost core was found to be dominated (94%) by a single genus, Sporichthya, an Actinobacteria which has been previously observed in sandstone cryptoendolithic communities in the Dry Valleys. This genus may represent a novel species as it has low similarity (<94%) to any sequence found on GenBank. Overall, our results suggest that the combination of subzero, arid, and very old UV permafrost is severely constraining microbial survivability, indicating UV permafrost is one of the most extreme cryoenvironments on Earth where viable microbial life is either severely limited or potentially nonexistent.

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103A Microbial Community Diversity and Their Roles on Nitrogen Cycling at Terrestrial Hydrothermal Systems Yasemin Gulecal*1, D'Arcy Meyer-Dombard2 1Istanbul University, Turkey, 2University of Illinois at Chicago, United States

Of all the biogeochemical cycles, nitrogen is the one most intimately and thoroughly associated with microbes. Essential and unique steps in the nitrogen cycle are performed by a wide array of bacteria, archaea, and eukaryotes, and the broad outlines of the cycle have been understood for over a century. Scientists have a little knowledge about nitrogen cycling at areas of hydrothermal systems. The objective of the study is to determine microbial community diversity and the roles of microorganisms on nitrogen cycling through genetic methods in Turkey which is on the 7th grade in the world in means of geotermal resources richness.

Genomic DNA isolation has been made on the samples taken from hot springs of Pamukkale, Karahayıt, Inalti, Tekke, Babacık, Hisarköy, Yalova and Koza. By using spesific primers, 16S rRNA gene and the genes taking part in nitrogen cycle nirS, narG, nifH and amoA bacteria and amoA archea amplified with PCR. These genes have been carried out with Sanger method after cloning and phylogenetic trees made. Also these genes had taken accession numbers from NCBI. In order to determine microbial diversity, a new generation sequencing method is '454 pyrosequencing' used.

As a result of this study, for the first time in Turkey hydrothermal waters, it has been detected that microorganisms do actively take roles on nitrogen cycling and their diversity is high even though there are extreme habitats.

104A Sulfur cycling at submarine freshwater springs in the Dead Sea Stefan Haeusler*1, Danny Ionescu1, Yaniv Munwes2, Christian Lott3, Miriam Weber3, Lubos Polerecky1, Christian Quast1, Thorsten Dittmar4, Aharon Oren5, Jonathan B. Laronne2, Dirk de Beer1 1Max Planck Institute for Marine Microbiology, Germany, 2Ben Gurion University, Israel, 3HYDRA Institute for Marine Sciences, Italy, 4Carl von Ossietzky University, Germany, 5The Hebrew University of Jerusalem, Israel

The Dead Sea is a hypersaline lake with up to 350 g/l total dissolved salts. High concentrations of Mg (2 M) and Ca (0.5 M) make it a hostile environment where only unicellular organisms can survive. Recently discovered underwater freshwater springs in the Dead Sea show a high potential to be islands of life in this ecosystem.

We investigated a thin white microbial mat in a zone of slow advective freshwater flow through the Dead Sea sediment. Pyrosequencing and Fluorescence in situ hybridization showed a dominance of sulfide oxidizing epsilonproteobacteria and gammaproteobacteria as well as sulfate reducing deltaproteobacteria suggesting an active sulfur cycle. In situ microsensor measurements confirm the consumption of oxygen and sulfide in the active zone where the mat was located. Incubations using 13C labeled bicarbonate and acetate showed the presence of both autotrophic and heterotrophic activity; however, the uptake of acetate was higher suggesting chemolithoheterotrophy is a central metabolic pathway. Sulfate reduction was measured using radiolabeled sulfate and was found to be in the range of known marine environments. The system seems therefore to depend on supply of dissolved organic matter which is abundant in the spring water. Although the latter also deliver 15 µM of sulfide, the community might not necessarily depend on an external source since recycling of sulfur species can occur between the sulfate reducing and the sulfide oxidizing bacteria. This is the first description of an active sulfur cycle in the Dead Sea.

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105A Meta-transcriptomic analysis of microbial community associated with a soda lake in Khovsgol, Mongolia Natsuko Hamamura*1, He Huang2, Shujiro Okuda3, Narantuya Damdinsuren4 1Ehime University, Japan, 2Center for Marine Environmental Studies (CMES), Ehime University, Japan, 3Department of Bioinformatics, Ritsumeikan University, Japan, 4School of Biology and Biotechnology, National University of Mongolia, Mongolia

Soda lakes are extreme habitats that have a stable high pH and high salt content, and are occasionally associated with elevated concentrations of trace elements from volcanic origins, including arsenic (As). To gain insight regarding the ecophysiology of microbial populations associated with such an extreme environment, meta-transcriptomic analysis was applied to detect community gene expressions in a soda lake sediment from Tokh Lake, Khovsgol, Mongolia, before and after exposure to arsenate (10 mM). cDNA libraries of amplified RNA from the soda lake sediments were sequenced with Illumina HiSeq at a depth of ~1Gbp each. Majority of the obtained sequences (80~84%) were identified as rRNAs by screening against small and large subunit rRNA databases. The remaining sequences were then compared against an NCBI non-redundant protein database using BLASTX (E-value <10-5) and identified as predicated mRNA sequences (1.3~1.7% of total sequence reads). Community taxonomic compositions of rRNA sequences indicated that the members of gamma-Proteobacteria, including Halomonas, Vibrio, and Pseudomonas spp., were most dominantly present before the exposure. Identification of highly expressed functional groups revealed that the genes involved in general cellular functions (e.g. protein, carbohydrate, RNA metabolisms) were abundantly expressed in both transcriptomes and consisted of 40~45% of all the predicted mRNAs with assigned functions. After the exposure of the lake sediment to arsenate for 30 min, community taxonomic compositions of rRNA exhibited increases in the relative abundance of Vibrio spp. and increases in expression of genes associated with virulence and sulfur metabolism functions. Specifically, those transcripts were assigned to arsenic resistance genes including arsenate reductase and thioredoxin. Thioredoxin has been shown to catalyze the reduction of wide range of target proteins including arsenate reductase (ArsC), a key enzyme involved in arsenic detoxification. Exposure to arsenate led to reduction in transcripts associated with nitrogen fixation, nitrate reductase, and nitrate transport. Our results demonstrate the potential application of meta-transcriptomics to elucidating community-level functionality and response of indigenous microorganisms associated with a soda lake environment.

106A Deep sea hydrothermal vent enrichment cultures: A way to find novel hydrogenases Moritz Hansen*, Mirjam Perner University of Hamburg, Germany

At deep sea hydrothermal vents primary biomass production is largely fueled by microbial oxidation of reduced inorganic compounds, for example hydrogen. These inorganic substrates are transported by the hydrothermal fluids from inner earth. Mixing processes between hot, highly reduced hydrothermal fluids and cold, oxygenated ambient seawater cause steep physical and chemical gradients in the hydrothermally influenced habitats, which can be colonized by phylogenetically and physiologically diverse hydrogen oxidizers. For conversion of hydrogen these microbes have key enzymes, namely hydrogenases, which catalyze the reversible reaction of H2 ↔ 2H+ + 2e-. Since these hydrothermally influenced provinces can range in their physico/chemical properties from anoxic to fully oxic and extremely hot (350 °C) to cold (4 °C), they represent ideal systems for harnessing hydrogenases hallmarked by properties relevant for industrial applications. For example, for fuel cells or electrochemical cells, hydrogenases that are oxygen tolerant are desired.

Here we describe an enrichment culture with autotrophic hydrogen oxidizers, which was inoculated with low-temperature hydrothermal fluids from Sisters Peak (5° S on the Mid-Atlantic Ridge). Cultivation is performed in artificial seawater with bicarbonate under an atmosphere of 79% hydrogen, 20% carbon dioxide and 1% oxygen at 28 °C. We have analyzed the community compositions and investigated the hydrogenases expressed in this enrichment.

To characterize the cultured community, 16S rRNA genes were amplified and fluorescence in situ hybridizations with species-specific probes were performed. Uptake hydrogenase activities were determined spectrophotometrically by following the reduction of methyl viologen. Hydrogen uptake measurements were monitored using gas chromatography.

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Analyses of 16S rRNA genes revealed the presence of four different species in this culture, namely the Gamma-Proteobacteria Thiomicrospira crunogena, Alteromonas macleodii, Pseudomonas pachastrellae and the Alpha-Proteobacterium Thalassospira sp.. Fluorescence in situ hybridizations illustrated clear shifts in the culture community over time. The activity of membrane associated unpurified proteins isolated from the enrichment was at least 0.25 ± 0.07 µmol H2*min-1*mg-1, while the soluble unpurified proteins exhibited only low activity (0.0006 ± 0.03 µmol H2*min-1*mg-1). Hydrogen consumption rates clearly demonstrated the active biological uptake of hydrogen during growth of the culture. To identify the hydrogenase expressing species we pursue 2 strategies: (i) investigations of the isolated species with respect to hydrogen oxidation and (ii) sequencing of active hydrogenases separated from other proteins via Native PAGE and identification by methyl viologen reduction.

The investigated mixed culture grows autotrophically with hydrogen as sole energy source. Interestingly, none of the identified species are known to utilize hydrogen as sole energy source and only T. crunogena is known to grow autotrophically. However, based on uptake hydrogenase activity of membrane associated proteins and hydrogen consumption rates the results evidence that active hydrogenases are being expressed by the mixed culture. Since the species do not grow as isolates with hydrogen as only electron donor, we assume interactions between the species regarding the oxidation of H2 and CO2 fixation in the mixed culture.

107A Comparison of microbial assemblages in Arctic multi-year and first year sea ice Ido Hatam*, Brian Lanoil, Christian Haas University of Alberta, Canada

Arctic sea ice is a vast habitat that supports a microbial ecosystem that is active year round and is a significant component of biogeochemical cycling and energy flux in the Arctic Ocean. Until recently, the Arctic was dominated by multi-year ice (MYI; ice that survived at least one summer); however, MYI levels have dwindled to just 45% of the total ice cover over the past 30 years due to climate change. Furthermore, it is predicted that by the end of the century, MYI cover will be completely gone. The remaining ice is now predominantly first-year ice (FYI; new ice that melts in summer), which is younger, thinner and less stratified in terms of salinity, nutrients and temperature than MYI. We tested the hypothesis that due the physical and chemical differences between FYI and MYI; the two types of ice hold different microbial assemblages. In mid-spring 2010 and 2011, we obtained both MYI and FYI cores as well as underlying sea water from the Arctic Ocean, north of the Canadian Forces Station Alert; Ellesmere Island, Nunavut. As expected, the MYI showed an increase in salinity and pH values as depth increased, whereas these values were more homogenous in FYI cores. Denaturing gradient gel electrophoresis (DGGE) of the V3 region of the 16S rRNA gene showed that bacterial assemblages clustered based on ice type (MYI versus FYI). Furthermore, a depth profile of the microbial community structure of both types of ice demonstrated a clear change in assemblage composition for MYI sub-sections but not FYI sub-sections. This may reflect a distinct and yet unrecognized ecological role for different layers of MYI, one that may not be performed by the assemblages in FYI. In order to confirm these preliminary results, we are characterizing both assemblages using Roche 454 pyrosequencing and the 16S rRNA gene V1-V3 regions as markers. We are currently in the process of analyzing a 100,000 sequence data. If these findings support distinct assemblages both between different layers of MYI and between FYI and MYI, it will help predict and understand the likely response of the Arctic Ocean to climate change over the next 50 years.

108A Unrevealed culturable bacterial diversity in Lirima wetland, a high Andean sulfurous hot spring Martha Hengst*1, Klaudia Hernandez2, Veronica Molina3, Giannina Maya4, Cristina Dorador1 1Universidad de Antofagasta, Chile, 2CIEN Austral, Universidad Austral, Chile, 3Universidad Andres Bello, Chile, 4Universidad de Concepcion, Chile

Lirima wetland, is located a high altitude (4.200 m above sea level) which belongs to the Chilean Altiplano, northern Chile (19°51.110'S; 68°54.400'W). This ecosystem is characterized by the presence of freshwater hot-springs of a wide temperature range (from 19°C to 89°C), which is originated from groundwater sources, conforming imbricate channels network, dominated by thermophilic microbial mats highly pigmented. Extreme weather conditions are typical in this location such as high values of total solar radiation (>1000 Wm-2), a strong environmental thermal oscillation

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~40°C (day/night), besides low oxygen pressure and a negative water balance. All of these variables contribute to generate a suitable environment for the growth and development of particular microbial communities which had not been previously obtained in pure culture. Our aim was to characterize culturable pigmented bacterial diversity from Lirima wetland in selective media enriched in sulfur compounds, and to detect the potential as source of bioactive natural compounds. The identification of the isolates was using 16S rRNA gene as molecular marker and the potential capability to produce bioactive substances was detected through PCR amplification of Polyketide synthase (PKS-I) and non-ribosomal peptide synthetase (NRPS) genes. The results showed a total of 61 bacterial isolates that were obtained in pure cultures, 35 of them were strongly pigmented (orange, brownish, and red) and the remaining 26 were colorless or no pigmented. The diversity of bacteria was distributed in 18 genera, belonged to Actinomycetes (3%), Bacteroidetes (3%), Firmicutes (37%), Alpha- (14%) and Gamma-Proteobacteria (43%). Moreover, our results showed that 33% of the total isolates from Lirima wetland posses at least one gene cluster encoding for nrps genes, involved in the synthesis of bioactive substances. Several bacterial isolates obtaining in Bofedal Lirima are also reported in another extreme habitats, notably cold, saline and hydrothermal environments, as Artic marine ice, glaciers, and hypersaline lakes. Our findings highlight how that unique environmental conditions of high altitude, irradiation, and daily thermal fluctuation are important factors in the selection of pigmented microbial communities of Lirima wetland.

109A The physiological characterisation of a novel Thermomicrobia isolate Karen Houghton*1, Xochitl Morgan2, Kirill Lagutin3, Mikhail Vyssotskii3, Andrew MacKenzie3, Hugh Morgan1, Matthew Stott2 1University of Waikato, New Zealand, 2GNS Science, New Zealand, 3IRL, New Zealand

Thermomicrobia is a novel class within the phylum Chloroflexi that currently contains only two isolated species; Thermomicrobium roseum, isolated from Yellowstone National Park, USA, in 1973, and Sphaerobacter thermophilus, isolated from a wastewater plant in Germany in 1989. Here we present work that characterises the physiology and ecology of a third newly isolated strain from this group, WKT50.2.

WKT50.2 is an aerobic, thermophilic bacterium isolated from a geothermally-heated soil rich in degraded plant and organic matter, at Waikite, New Zealand. It is phylogenetically distinct from both T. roseum and S. thermophilus sharing 16S rRNA gene sequence similarity of only 91% and 88% respectively. However, all three strains share some similar traits including pink/red pigmentation, circumneutral growth pH, thermophily and a primarily proteolytic metabolism. Surprisingly, we have shown that WKT50.2 also grows on a range of cellulolosic substrates including carboxymethylcellulose, crystalline cellulose (Avicel), wood pulp and xylan. This observation reflects the ecological niche in which WKT50.2 was isolated, but differs with the reported metabolic capabilities of T. roseum and S. thermophilus. We also define the lipid composition and the temperature and pH growth ranges for WKT50.2.

110A Comparative Metagenomics Along a Thaw Gradient of Alaskan Permafrost Jenni Hultman*1, Maude M. David2, Olivia U. Mason2, Regina Lamendella2, Manesh B. Shah3, Nathan Verberkmoes3, Mark P. Waldrop4, Janet K. Jansson2 1University of Helsinki, Finland, 2Lawrence Berkeley National Laboratory, United States, 3Oak Ridge National Laboratory, United States, 4United States Geological Survey, United States

The fate of organic carbon reserves sequestered in permafrost is uncertain yet critically important for addressing terrestrial feedbacks to climate change. With warming there is an increased probability of thermokarst formation, and an increase in CO2 and CH4 flux to the atmosphere. However, we understand little of the underlying microbial controls on nitrogen or carbon cycling in permafrost soils. We applied a variety of “omics” methods to study microbial communities, their functions and activity in permafrost soils collected from two sites in Alaska: a stable, low productivity black spruce forest, and a thermokarst bog at the Bonanza Creek LTER Station outside of Fairbanks. Permafrost samples were taken from both the active and the permafrost layers. Our aim was to understand microbial functions in the longer-term thaw processes that are ongoing in the arctic. In total, over 170,000 pyrotags were sequenced and >40 Gb of shotgun metagenome data was obtained. Additionally, shotgun metatranscriptomic and metaproteome approaches were used to examine expression patterns in the different samples. The pyrotag results indicated that the bacterial communities in the permafrost and

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bog samples were more similar to each other than to the active layer. The bog samples had a different archaeal community compared to the other sample types, with a predominance of methanogens. This is consistent with the bog environment being an area of high CH4 production. An uncultured representative of the Chloroflexi phylum was found to be cosmopolitan and highly abundant (up to 15% of total pyrotag reads) in all sites. When looking at the metagenome gene content, bog and permafrost samples were more similar. There were more differences between the replicate cores in bog than in permafrost and active layers, indicative of greater functional differences in the bog site. We binned the metagenomic contigs based on tetranucelotide frequency, and taxonomic assignment of the largest bins were to uncultured Deltaproteobacteria and Actinobacteria. Microbes from these groups were also found to be active based on the metatranscriptomic sequencing. Metaproteomes were analyzed from 6 samples resulting in close to 800 protein IDs in active layer samples and 450 in permafrost. Analysis of the metaproteomes revealed methanogenesis to be occurring in thermokarst bog, and methane to be actively oxidized in the active layer. In the permafrost samples (depth 70 cm) the expressed proteins were involved in membrane transport and sugar transport. Overall we found that permafrost soils harbor unique microbial communities that do not contain the diversity of archaea present in thermokarst bogs. Therefore important shifts in community composition occur following thaw and before the initiation of methane production.

111A Culture-independent bacterial monitoring with adhesive sheet in the "Kibo", a part of the International Space Station Tomoaki Ichijo*, Hatsuki Hieda, Rie Ishihara, Nobuyasu Yamaguchi, Masao Nasu Osaka University, Japan

The International Space Station (ISS) has been staffed continuously since the first resident crew entered the facility on 2 November 2000, thereby providing a permanent human presence in space. We have been continuously monitoring the abundance of bacteria and predicting their dynamics in the "Kibo", a part of the ISS (project title: MICROBE). In this project, we use a new sampling device, the microbe-collecting adhesive sheet, which was developed in our laboratory. This adhesive sheet has high operability, needs no water for sampling, and is easy to transport and store. In the present study, prior to sampling in the Kibo, we demonstrated the applicability of the adhesive sheet as a sampling device in the Kibo, with regard to stability of bacterial cell number during prolonged storage of up to 12 months. We then used the adhesive sheet for bacterial monitoring in the Kibo.

For evaluating the applicability of an adhesive sheet, cultured bacterial cells (mixture of Acinetobacter lwoffii, Bacillus subtilis, Pseudomonas putida, Staphylococcus epidermidis) were collected on the sheet and stored at room temperature (approx. 20°C), 4°C or -80°C until up to 12 months. Then, changes in bacterial number on the sheet during prolonged storage at various temperatures were investigated with fluorescent staining. On 29 October 2010, samples were taken by applying the adhesive sheets to the interior surfaces and equipment in the Kibo. After the samples were transported to the laboratory, bacterial abundance and taxonomic distribution were determined.

The number of bacterial cells on adhesive sheets stored at room temperature and 4°C was markedly decreased during storage. In contrast, the number of bacteria collected on the adhesive sheet and stored at -80°C was >90% and it did not significantly change during this period (until 12 months) (P < 0.05; Student's t test). Therefore, after storage of samples at -80°C, the counts obtained reflect the abundance of bacteria collected on the day of sampling (time zero). We then used the adhesive sheet for bacterial monitoring in the Kibo. The abundance of bacteria in the Kibo determined by culture-independent approaches (fluorescent staining and quantitative PCR) was lower than that on the surfaces in our laboratory (105 cells/cm2). Taxonomic distribution of bacteria collected from surfaces of the four sites were determined by PCR-DGGE followed by sequencing. Bacteria in the phyla Actinobacteria and Firmicutes were frequently detected on the surfaces which astronauts contact frequently. These phyla have been reported as the dominant ones of the human skin microbiome; thus, bacterial cells might be transferred to the surface in the Kibo via astronaut contact.

The Kibo has been microbiologically well maintained; however, microbial abundance may increase with prolonged stay of astronauts. To ensure crew safety and understand bacterial dynamics in space habitation environments, continuous bacterial monitoring in the Kibo is required.

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112A Genome analysis of Haloarchaea isolated from seawater Kentaro Inoue*1, Kenshiro Oshima2, Mikihiko Kawai3, Hideto Takami4, Masahira Hattori2, Kazuhiro Kogure1 1University of Tokyo, Atmosphere and Ocean Research Institute, Japan, 2University of Tokyo, Center for Omics and Bioinformatics, Japan, 3JAMSTEC, Environmental Metagenome Research Team, Japan, 4JAMSTEC, Microbial Genome Research Group, Japan

Haloarchaea are extremely halophilic, aerobic members of Archaea, classified within the family Halobacteriaceae. Recently, we succeeded to isolate haloarchaea from multiple seawater columns and also to detect their presence by culture-independent methods. Our result indicates that haloarchaea belonging to variety of phylogenetic group are commonly present in marine environments This is contradictory to the general view that seawater salinity (circa 3.5%) is too low for normal maintenance of the cell wall. Many questions remain to be answered; when and which group entered the sea? Where they entered and how to adapt to lower salinity environments? The purpose of this study was to answer a part of these questions, especially adaptation mechanism by genome sequencing analysis.

Whole genome sequencing analysis of Halomarina oriensis KeC-11T, isolated from seawater aquarium, was carried out. Followed by gene annotation using the BLAST (Basic local alignment search tool) search and the Clusters of Orthologous Groups (COGs) of proteins, comparative analysis of complete haloarchaeal protein sets were carried out.

The genome of strain KeC-11T is 4,072,440 bp long and comprises one main circular chromosome of 3.1 Mbp with a 67.7% G+C content and nine plasmids that have a total of 0.96 Mb with 59.1 to 67.9% G+C contents. As the adaptation mechanism, first, the presence of multiple plasmid possession is presumably a result of acquiring genes for adaptability in seawater. Second, it has osmoprotectant transport pathway which is prevailing mechanism among only other moderate halophiles. Therefore, H. oriensis uses both “organic-solutes-in strategy” and “high salt-in strategy” strategies to re-establish turgor pressure and to circumvent the change of salinity. Because the “organic-solutes-in strategy” is rare among haloarchaea, it is assumed that horizontal gene transfer occurred prior to entering marine environments.

It is concluded that three factors, that is gene transfer events, acquiring gene for osmoadaptation, and utilization of the two strategies, “high-salt-in cytoplasm strategy” and “organic-solutes-in cytoplasm strategy” made them possible to enter seawater environments. It is assumed that the gene transfer events occurred multiple times among haloarchaea belonging to different phylogenetic groups.

113A Springs of life in a Dead Sea - rich microbial communities in and around a complex system of underwater springs in the Dead Sea Danny Ionescu*1, Christian Siebert2, Stefan Häusler1, Thorsten Dittmar3, Lubos Polerecky1, Yaniv Munwez4, Christian Lott5, Miriam Weber5, Mina Bižić-Ionescu6, Christian Quast1, Aharon Oren7, Stefan Geyer2, Jonathan B Laronne4, Dirk De Beer1 1Max Planck Institute for Marine Microbiology, Germany, 2Helmholtz-Centre for Environmental Research – UFZ, Germany, 3Max Planck Institute for Marine Microbiology, Carl von Ossietzky University, Institute for Chemistry and Biology of the Marine Environment (ICBM), Germany, 4Ben Gurion University, Israel, 5Max Planck Institute for Marine Microbiology, HYDRA Institute for Marine Sciences, Germany, 6Max Planck Institute for Marine Microbiology, Leibniz Institute of Freshwater Ecology and Inland Fisheries Berlin (IGB), Germany, 7The Hebrew University of Jerusalem, Israel

The Dead Sea is a terminal, hypersaline desert-lake on the border between Jordan, the Palestinian Authority and Israel. In the last decades, due to reduced freshwater water inflow and high evaporation the salinity of the lake reached ~350 g/l total dissolved salts, and the water level has been decreasing by over 1 m per year. The waters of the Dead Sea contain 2 M Mg and 0.5 M Ca thus being toxic to most life forms. The sole known primary producer in the lake, Dunalliella sp., appears only after massive dilution events during heavy winters. Generally the Dead Sea is characterized by very low cell densities, most of which are Archaea. We discovered several systems of underwater fresh to brackish water springs in the Dead Sea, often located at depths that did not allow detection from the shore. We explored the waters and outflow area of one such system. The water contained up to 107 cells, higher

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than normally described for non-polluted groundwater. Community analysis and water chemistry suggest that sulfate reduction and sulfide oxidation occur in the underground system. Furthermore, coupled with fluorescent in situ hybridization, they suggest that there is a large archaeal community in the shoreline pore water of the lake. Dissolved organic carbon fingerprinting using electrospray ionization Fourier transformation cyclotron resonance mass spectrometry detected masses of ~20,000 different compounds. Molecular formulae could be assigned to ~5000 of them. The nature of the organic matter suggests most of it has been microbially transformed. Peptides or compounds deriving from higher plants were not detected. Furthermore, evidence of thermogenic organic matter is found. All these may suggest that the heterotrophic system in the underground water system is fueled by fossil organic matter. In the area of the spring outflow, dense microbial communities were found including biofilms of diatoms and phototrophic and non-phototrophic sulfide oxidizers. Pyrosequencing of the 16S rRNA gene and fingerprinting methods showed that the spring- sediment community originates from the Dead Sea sediments and not from the spring water-borne community. Sequences of bacterial sulfate reducers, nitrifiers, iron oxidizers and iron reducers were identified as well. Analysis of green and white biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant in these regions. Hyperspectral analysis additionally showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which are not known from other hypersaline environments. The mechanisms which allow these organisms to survive the harsh conditions of the Dead Sea until favorable conditions, such as found in the freshwater springs, are encountered are still unknown. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of the possible impact of dilution events of the lake surface waters, such as those that will occur if the intended Red Sea-Dead Sea water conduit is to be established.

113B Microbial life in a geothermal fluid Marion Jaussi*1, Nicole Jeanneret1, Sevasti Filippidou1, Thomas Junier1, Tina Wunderlin1, Ludovic Roussel-Delif1, Andrea Vieth-Hillebrand2, Simona Regenspurg2, Pilar Junier1 1University of Neuchâtel, Institute of Biology, Switzerland, 2Helmholtz-Zentrum Potsdam, Deutsches GeoForschungsZentrum GFZ, Germany

Environmental conditions in geothermal fluids from deep aquifers suit the definition of an extreme environment: high pressure, elevated temperatures (up to several hundred degrees Celsius), high salinity and anoxia. Even under such harsh conditions microbial life is present. This research project is the first genotypic and phenotypic characterization of microorganisms isolated from deep geothermal reservoirs.

Samples of geothermal fluid (50-70°C) and deposits from filter bags were collected during the conditioning of the thermal water loop at the geothermal laboratory of Groß Schönebeck (Germany). Enrichment and isolation of bacteria were performed using adapted media and under in situ pH and temperature conditions. Two endospore-forming strains (GS3372, GSsed3) were isolated from the geothermal fluid. Based on the 16S rRNA gene sequence, these isolates were affiliated to the genus Geobacillus of the phylum Firmicutes. The closest cultured relatives to strains GS3372 and GSsed3 were Geobacillus pallidus (Aeribacillus pallidus) and Geobacillus stearothermophilus, respectively. Morphological description, physiological analysis and biochemical characterization were undertaken according to the minimal standards for the description of new taxa of aerobic, endospore-forming bacteria. Reference strains G. pallidus DSM 3670T and G. stearothermophilus DSM 22T were analyzed for direct comparison to the obtained isolates. The morphological description of the new strains was similar to their respective reference strain. However, a number of physiological and biochemical differences were observed. For example, the strain GS3372 was not mobile and used fewer carbon sources than strain DSM 3670T. Strain GSsed3 showed urea hydrolysis and no reduction of nitrates unlike DSM 22T. In general, the isolates are more selective in respect to nutrient utilization than the reference strains.

The new isolates were well adapted to the conditions of the geothermal fluid but appear to be less suited for the conditions in the deep reservoir. Indeed both strains do not grow at temperatures higher than 65°C. Their salinity tolerance was considerably lower (30-50 g/L) than the measured salinity in Groß Schönebeck fluid (190-260 g/L).

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Endospores are well known for survival under unfavourable conditions and for long periods of time. Accordingly, we hypothesize that these organisms are present in the form of endospores in the hot, deep aquifer. Upon decrease of fluid temperature in the geothermal pipes or upon cultivation the endospores are revived. Unequal evolutionary pressures might have caused physiological differences between isolates and the reference strains

114A Survival of microorganisms to the main stress factors encountered in clouds Muriel Joly*1, Pierre Amato2, Martine Sancelme2, Mickael Vaitilingom2, Virginie Vinatier2, Laurent Deguillaume3, Anne-Marie Delort2 1Blaise Pacal University - Institut de Chimie de Clermont Ferrand - Laboratoire de Météorologie Physique, France, 2Blaise Pacal University - Institut de Chimie de Clermont Ferrand, France, 3Blaise Pacal University - Laboratoire de Météorologie Physique, France

Microorganisms are present in low altitude clouds at concentrations of ~104 bacteria per mL and ~103 fungi per mL. The microbiological monitoring of cloud water since 2003 at the puy de Dôme station (1465 m a.s.l., France) revealed the presence of recurrent microorganisms among the cultivable bacterial community: Pseudomonas spp. and Sphingomonas spp. notably were present in 55% and 45% of the samples collected respectively, and they represented 30% of the total heterotrophic bacteria cultivable at 17°C.

Clouds represent nearly obligate ways to the redeposition of micrometer-sized aerosols such as microorganisms onto the ground. Even though they are believed to be less aggressive environments than dry air, clouds likely remain harsh and probably operate as environmental filters to airborne microorganisms through various selection factors: solar radiation, oxidants, low temperature, acidity, osmotic shocks, etcetera.

In order to test this hypothesis, we selected five bacterial or yeast strains isolated from cloud water (Arthrobacter sp. AJ551167; Pseudomonas syringae AB001440 and HQ256872; Sphingomonas sp. HQ256831; Bullera armenica JF706549). These strains were chosen to offer a good representation of the particularities of the culture collection (Gram-positive and Gram-negative bacteria, pigmented or not, ice nucleation active or not). In parallel, an Escherichia coli strain (ATCC10798) was chosen as reference as none was found viable among our cloud samples.

The survival rates of each of these strains to four factors of stress consistent with the conditions encountered in clouds were investigated: osmotic shock, which occurrs when water vapour condenses or evaporates around airborne microorganisms, freeze-thaw cycles, presence of toxic compounds such as hydrogen peroxide, and solar radiation. The first results demonstrated that the strains originally isolated from clouds were not particularly resistant to these four treatments, compared with E. coli. Hence those selective factors are not sufficient to explain the composition of the viable community in clouds.

115A Extreme plasticity of biological stoichiometry Carriayne Jones*1, Sean Crowe1, Julia Maresca2, Edward DeLong3, Bernhard Viehweger4, Kai-Uwe Hinrichs4, David Fowle5, Sulung Nomosatryo6, Donald Canfield1 1University of Southern Denmark, Denmark, 2University of Delaware, USA, 3Massachusetts Institute of Technology, USA, 4University of Bremen, Germany, 5University of Kansas, USA, 6Indonesian Institute for Sciences, Indonesia

Phosphorus is an essential nutrient for life, and aquatic microorganisms are generally believed to require a fixed quantity of phosphorus for growth. This is reflected in the stoichiometric relationships between C, N, and P typically observed in the nutrient pools and biomass of aquatic environments (the Redfield Ratio- 106C:16N:1P). Recent reports, however, suggest this stoichiometry may be more flexible than previously thought. Here, we show the extent of this flexibility in the microbial community of Lake Matano, an ultraoligotrophic lake on Sulawesi Island, Indonesia. Local geology, mostly primitive mantle rocks, weathers to produce soils rich in Fe oxides, which strongly sorb P from meteoric, ground, and lake water. This results in extremely low dissolved P concentrations in the surface waters of Lake Matano.

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Based on nutrient concentrations, elemental composition of biomass, alkaline phosphatase activity (APA), C-fixation assays, metagenomics, and lipids in microbial biomass, we show that P limits production in the surface waters. Furthermore, the extreme scarcity of P has led to exceptionally high bulk C:P ratios (>1000) in plankton, more than an order of magnitude higher than the Redfield ratio. Slow growth rates of microbes imply that, to achieve such low P cell quotas, they are minimizing P usage by limiting their RNA requirements. Additionally, no phospholipids have yet been detected in the microbial biomass, suggesting P-free glycerolipids and glycolipids substitute for phospholipids in cell membranes. We have detected genes involved in sulphoquinovosyldiacylglycerol biosynthesis (SqdX), which are known to play a role in phytoplankton growth strategies under P limiting conditions in the ocean. High APA and no detectable dissolved organic P suggest rapid P turnover rates. The detection of Pho genes, which code for proteins utilized in both inorganic P scavenging and uptake of alternative P sources (e.g. organophosphorus and phosphonates) indicates a variety of strategies for P acquisition. C-fixation was stimulated with P additions, but the C-fixation rates did not increase proportionally with the amount of P added, suggesting some of the assimilated P was either stored for future use or used by heterotrophs.

Together, our data show that microbes in Lake Matano's surface waters are under severe P stress. Beyond these microbial ramifications, this P stress translates to very low lake productivity. The low P microbial community provides poor nutrient quality at the base of the food web, which is a possible cause for the truncated trophic pyramid in the ecosystem, explaining a conspicuous absence of piscivores and other tertiary consumers.

116A Diversity and distribution of cyanobacteria and microbialites in lakes of the McMurdo Dry Valleys, Antarctica Anne Jungblut*1, Ian Hawes2, Dawn Y Sumner3, Tyler Mackey3, Lu Zhang1, Dale T. Andersen4 1The Natural History Museum, United Kingdom, 2University of Canterbury, United Kingdom, 3University of California, USA, 4Carl Sagan Center for the Study of Life in the Universe, USA

Mat-forming cyanobacteria are among the oldest known prokaryotes and are widely distributed in more extreme environments, where grazers are absent or marginalized because of extremes in temperature, salinity, and UV. The physical form of the cyanobacteria living within the mats and the three dimensional structure of mats themselves is very conserved with fossils dating from more than 2 billion years before the present similar to living mats. However, given the wide variety of habitats where mats tend to be abundant a major question is the extent to which, within and between environments, morphological similarity of structures reflects the presence of similar genotypes, and whether particular genetic adaptations or metabolic pathways common to different organisms may be in place that facilitate specific emergent morphologies.

In the McMurdo DryValleys, Antarctica, there are several perennially ice-covered meromictic lakes including Lake Vanda, Joyce and Hoare, which differ in their chemical and physical properties. The biology of these lakes is mostly microbial and thick, stratified cyanobacteria-based mats and microbialites are common features of these unique Antarctic aquatic ecosystems. We will describe prostate mats and microbialites with some of them having discrete layer and cyanobacterial diversity found in these lakes along vertical gradients using morphological descriptions, and 16S rRNA gene surveys.

Although the impacts of environmental change have mostly been documented from maritime Antarctica, recent findings have confirmed overall warming of continental Antarctica and sensitivity of inland aquatic systems to such change. We will focus on evidence of change in lakes of the McMurdo Dry Valleys, examine how microbialites and cyanobacterial diversity have responded to variable water dynamics and look at the implications of change for Antarctic aquatic inland ecosystems.

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117A High-energy geochemistry drives microbial diversity and distribution in Yellowstone Lake Jinjun Kan*1, Scott Clingenpeel2, Charlie Dow1, Richard Macur3, William Inskeep3, Dave Lovalvo4, John Varley3, Yuri Gorby5, Timothy McDermott3, Kenneth Nealson5 1Stroud Water Research Center, USA, 2DOE Joint Genome Institute, USA, 3Montana State University, USA, 4Eastern Oceanics, USA, 5Univeristy of Southern California, USA

Yellowstone Lake (Yellowstone National Park, WY, USA) straddles the most recent Yellowstone caldera, and is situated on top of significant hydrothermal activity. In order to evaluate how the hydrothermal vents influence the biology of this young freshwater ecosystem, geochemical and microbiological characterizations were applied to samples obtained by a remotely operated vehicle in 2007 and 2008, including water directly from the hydrothermal vents as well as lake surface. The temperatures observed in hydrothermal vents ranged from 40-94°C, and elevated concentrations of H2S, CH4, CO2 and H2 were observed. Biomass was size-fractionated by sequentially filtering through 20µm-, 3.0µm-, 0.8µm-, and 0.1µm-pore-size filters. Bacterial and Archaeal population structures were assessed by using 454-FLX pyrosequencing and Sanger sequencing of 16S rRNA gene. In general, Acidobacteria, Actinobacteria (Freshwater acI), Aquificae, Bacteroidetes, Cyanobacteria (Prochlorococcus), Firmicutes, Alpha- (SAR11-like) and Beta-Proteobacteria, and Verrucomicrobia were the major bacterial phyla in both surface and vent waters. However, most of the archaeal diversity was associated with vents: Euryarchaeota (Deep Sea Euryarchaeal Group or Deep Sea Hydrothermal Vent Group-6) and Crenarchaeota (Marine Group 1, Thaumarchaeota) dominated archaeal groups. Vent waters contained thermophilic bacteria and archaea such as Aquificae, Caldiserica, Deinococcus-Thermus, OP10, OP11, Thermodesulfobacteria, Thermotogae, and Thermoprotei. The H2 levels were significantly higher than the reported growth threshold concentrations for hydrogenotrophs and high concentrations of methane suggested another potential energy and carbon source for the lake. Sequence classification identified prominent methonogenic and methylotrophic signatures including Archaea (Methanobacteria, Methanococci, Methanomicrobia) and Bacteria (Methylocystis, Methylobacillus, Methylotenera, Methylobacter, Methylibium). NMDS (Nonmetric Multidimensional Scaling) patterns showed that distinct bacterial population structures occurred among size fractions, suggesting that the fraction of >0.1 to 2, H2, CH4, H2S and CO2), DOC, NH4

+ and NO3-. In summary, rich microbial diversity and corresponding distribution patterns contradicts

the previous view that “Yellowstone Lake is a cold temperature, low nutrient, low productivity, and simple ecosystem”. In addition to phototrophy, H2 and CH4 likely make major contributions to the food chain in Yellowstone Lake. Finally, the phylogenetic parallels of Yellowstone Lake archaea to marine microorganisms provide opportunities to examine interesting evolutionary tracks between freshwater and marine lineages.

118A Isolation and physiological characterization of butanol- and isobutanol-tolerant bacteria Manabu Kanno*, Taiki Katayama, Hideyuki Tamaki, Yasuo Mitani, Tomoyuki Hori, Takashi Narihiro, Naoki Morita, Isao Yumoto, Nobutada Kimura, Satoshi Hanada, Yoichi Kamagata National Institute of Advanced Industrial Science and Technology, Japan

Toxicity of butanol or isobutanol is one of the most profound issues in microbial production of these solvents as biofuels. Only a few bacterial species are able to tolerate more than 2.0% (vol/vol) butanol, although a number of bacteria tolerating other organic solvents such as toluene and benzene have been isolated from the environments. The aim of this study is to extensively explore microbes with superior butanol- or isobutanol-tolerance from various environments and to characterize their physiological traits associated with butanol tolerance. For enrichment of butanol- and isobutanol-tolerant microbes, environmental samples obtained from grease-contaminated soils, thermophilic anaerobic digester sludges and freshwater sediments were exposed to 2.0~9.0% (vol/vol) of butanol under aerobic or anaerobic conditions, which varied in duration from 2 days to 9 months. Eventually, we succeeded in isolating a total of 16 aerobic and obligatory anaerobic, mesophilic and thermophilic bacteria able to grow more than 2.0% (vol/vol) butanol and isobutanol. The isolates were phylogenetically diverse and widely distributed among at least seven genera, Bacillus, Lysinibacillus, Brevibacillus, Coprothermobacter, Enterococcus, and Hydrogenoanaerobacterium within the phylum Firmicutes and Cellulosimicrobium in the phylum Actinobacteria. Importantly, nine isolates except for three isolates belonging to the genus Enterococcus were distinct from the previously known butanol- and isobutanol-tolerant bacteria. Furthermore, four isolates were phylogenetically novel and showed relatively low 16S rRNA gene sequence similarities (<94%) to any other described species. The relatively high solvent-tolerant strains GK12 and CM4A, which were able to tolerate up to 3.0 and

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3.5% (vol/vol) butanol, respectively, were further characterized for their butanol tolerance properties. The obligatory anaerobic strain GK12, which was phylogenetically novel at the genus level within the family Erysipelotrichaceae, grew faster with than without butanol by successive subcultures with butanol, showing its adaptation ability. Although the untreated cells of strain GK12 increased acyl-chain length of membrane fatty acids in response to butanol exposure, such notable change was not observed in the adapted cells. The discrepancy indicates that the adapted cells of strain GK12 may improve butanol tolerance ability by unknown mechanisms different from the membrane modification, a well-known mechanism for butanol tolerance.Another strain CM4A, an aerobic bacterium closely related to Enterococcus faecalis, did not show adaptation to butanol but exhibited the highest tolerance among the isolates. In the presence of butanol, the strain CM4A increased the proportion of saturated and cyclopropane fatty acids in the cell membrane, which can serve for enhancing its butanol tolerance. Indeed, the mutants of Escherichia coli, overexpressing the cyclopropane fatty acid synthase gene derived from the strain CM4A enhanced the solvent tolerance. Strain CM4A also increased extracellular capsule structure in the presence of butanol, suggesting that this change would be also involved in butanol tolerance. In conclusions, this study clearly demonstrated that a wide variety of butanol- and isobutanol-tolerant microbes are in nature, and that those microbes could possess different physiological mechanisms (e.g. changes in membrane fatty acid composition and cell morphology) to tolerate high concentrations of butanol and isobutanol.

119A Evolution at fluctuating temperatures improves thermal tolerance, pre-adapts to novel environments and alters virulence of opportunistic pathogen Tarmo Ketola*1, Lauri Mikonranta1, Ji Zhang1, Kati Saarinen1, Anni-Maria Örmälä2, Johanna Mappes1, Jouni Laakso2 1University of Jyväskylä, Finland, 2University of Helsinki, Finland

Fluctuating temperatures have been suggested to select for generalist genotypes that are capable of performing well across range of temperatures. However, adaptation to thermal fluctuations could also increase or decrease the ability to inhabit novel environments. Especially the latter, pre-adaptation to a novel environment has recently been suggested to play a key role in species invasions to new environments and in the emergence of new pathogens. Although climatic scenarios suggest future increases in thermal fluctuations, there is a lack of experiments resolving if upcoming thermal fluctuations could cause evolution of thermal tolerance and furthermore evolution of invasive and pathogenic species. To test these ideas we set up a replicated evolution experiment where descendants of single Serratia marcescens clone were divided to 20 populations. Half of the populations were subjected to constant temperature (31 ºC), and in half of the populations temperatures fluctuated daily between two extreme temperatures (24, 38 ºC). After circa 1000 generations we extracted 10 clones per population and measured growth rates and maximal biomass of evolved clones (totaling 240 clones) in three different temperatures (24, 31, 38 ºC), two chemical stressors (H2O2, DTT) and with the presence of ciliate predator (Tetrahymena thermophila) or lytic bacteriophage (PVV, Podoviridae). Finally, we tested virulence of evolved clones against Drosophila melanogaster insect host. In accordance with the hypotheses, we found that clones that evolved in fluctuating temperature for ca. 1000 generations, outperformed bacteria that evolved in constant temperature. This occurred not only in all of the temperatures, but also in novel environments such as chemical stressors, parasitic viruses, and predators. However, strains from fluctuating environments were found to be less virulent in Drosophila melanogaster insect host. Our results support the theories of evolution of thermal generalism and general tolerance across several novel environments, but also indicate that pathogen’s performance outside the insect host can be traded off with virulence. These results corroborate the importance of thermal fluctuations not only dictating the breadth of thermal tolerance, but also on the emergence genotypes capable of growing well in novel environments.

120A Biodiversity of prokaryotic microflora associated with halophytes in Farasan islands shallow salt sites Mohammad Khiyami* King Abdulaziz City for Science and technology, Saudi Arabia

Farasan is the largest island of the Farasan Islands, in the Red Sea. It is located 50 km offshore from Jizan, the far southwestern part of Saudi Arabia. It is located at 16°42′21″N41°59′0″E / 16.70583°N 41.983333°E / 16.70583; 41.983333 coordinates. It is considered a habitat for halophytes.

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We studied the bacterial communities in the shallow salt sites associated with halophytes and their distribution with respect to different physico-chemical characteristics like salinity gradients and types of pollutants. Soil, sediment and water samples were analyzed for their mineral and salt composition. Results showed that water was particularly rich in sodium, potassium, phosphorus and boron. Soil and sediments samples contained very high levels of calcium and magnesium, and moderate level of barium, boron and strontium. Sodium, phosphorus and iron levels varied to a large extent from one sample to the other. Neutral to very alkaline pH were recorded. Farasan Islands consists of numerous ecosystems including extreme environments in which microbial diversity has not been yet characterized. Water samples provided limited bacterial isolation whereas sediment and soil samples led to the isolation of a total of 83 bacterial species from 45 samples, belonging to various taxonomic groups. The isolates were arranged in two main clusters. The first cluster included seven bacterial species (in two subgroups) and the second contained two bacterial species Vibrio natriegens and V. rotiferianus.

Depending on the medium and the temperature of isolation, it can be hypothesized that some species were present as dormant structures, while some others, isolated at pH 8 on a medium rich in Na and Ca, could be in a growing form adapted to alkaline and saline conditions. Extreme halophilic organisms, particularly archaea, were recovered from different sites, which contained up to 255 g/l NaCl, whereas halotolerant bacteria were isolated from three sampling sites, which contained NaCl concentrations ranging between 6 to 32 g/l.

121A Bacterial community change during biofilm development in Arctic marine environment Eun Hye Kim*1, Kyeung Hee Cho1, Tae-Kyung Kim2, Yung Mi Lee1, Jang-Cheon Cho3, Hong Kum Lee1, Soon Gyu Hong1 1Korea Polar Research Institute, South Korea, 2Korea Bioinformation Center, South Korea, 3Inha University, South Korea

Biofilm is an important life form of microorganisms formed in aquatic environments including oral cavity, water supply system, fresh water and marine environments. They affect human health and water quality. It also have important ecological functions in aquatic environments such as protection from environmental stress and efficient lysis of macromolecules. In this study, we analyzed bacterial community change during biofilm development in coastal area of Svalbard, Norway. Massive sequencing technology was applied to determine bacterial community structures of biofilm samples collected once a day for 15 days. The major phyla were Alphaproteobacteria (50.3%), Bacteroidetes (38.2%), and Gammaproteobacteria (7.8%). Twenty one major OTUs (>3%) clustered by 97% sequence similarity cutoff mostly belonged to Rhodobacterales (33.5%) of Alphaproteobacteria and Flavobacteriales (9.5%) of Bacteroidetes. The major OTUs occupied 50-60% of the whole community. They were assigned to the genera Roseovarius, Loktanella, Pelagicola, Polaribacter, Sulfitobacter, Hoeflea, Planktomarina, Arenicella, Pseudoalteromonas, and several candiatus genera. Some of them were major components in the early phase and the others were major in the late phase.

122A Evidence for methane oxidation over 74°C in hot springs of Kuril Islands Anna Kizilova*, Irina Kravchenko, Nikolay Pimenov Winogradsky Institute of Microbiology, Russia

Little is known about microbial methane oxidation at temperatures over 50 degrees Centigrade. At the moment known moderately thermophilic and thermophilic methanotrophs belong to three genera of Gammaproteobacteria and phylum Verrucomicrobia. Highest temperature, at which methane was oxidized by a methanotroph, was shown for Methylothermus thermalis, a thermophilic methane-oxidizing gammaproteobacterium with upper temperature growth limit of 72°C.

During the expedition in August 2011 samples of water and sediments were collected in hot springs of the Kunashir Island, belonging to the Kuril Island chain, located at the Far East of Russia. The springs were versatile – water temperature ranged from 45 to 99 degrees, and pH varied from 2 to 10.

Potential methane oxidation was tested by means of radioactively labeled methane. Samples were incubated with 14CH4 at different temperatures for 11 days. Following springs St1 (74°C), St3 (77°C), St5 (76.6°C), St6 (68.4°C), Ni1 (88°C), P4 (83°C), Gp3 (98°C), and Gp4 (67°C) were incubated with 14C-methane at 76°C. In these springs pH varied from 4.5 to 7.5. Samples from three springs (St1,

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St3, St6) demonstrated credible methane oxidation activity at this temperature. Samples from four other springs (St5, P4, Gp3 and Gp4) also oxidized methane, however, the activity was quite low, thus we will seek for additional evidence of methanotrophic presence in these springs. Sample from Ni1didn’t show any signs of methane oxidation activity.

To date, this is the first evidence of methane oxidation at temperatures exceeding 72°C.

123A Microbe-mineral interaction in an extreme arid and high saline mine tailings environment in northern Chile Hananeh Korehi*1, Marco Blöthe1, Maria Alexandrovna Sitnikova1, Dagmar Kock1, Bernhard Dold2, Axel Schippers1 1Federal Institute for Geosciences and Natural Resources, Germany, 2FCFM-Universidad de Chile, Departamento de Geología, Chile

Chañaral Bay located in the Atacama Desert in northern Chile is an extreme arid and high saline environment that represents a site for the investigation of microbe-mineral interactions in mine tailings. Over 220 Mt of tailings from copper mining activities, averaging 0.8 wt% pyrite were dumped on the beach of the city Chañaral and caused metal pollution. The shore line is now over one kilometre further out to sea than it was original and the tailings cover about 4 km2 surface area with about 10 - 15 meters thickness. The tailings are exposed to air which has resulted in a 70-188 cm thick low-pH (2.6 - 4) oxidation zone with formation of Fe-hydroxides and a liberation of divalent metal cations such as Cu2+, Ni2+, Na2+ and Zn2+. To identify and quantify microbial communities relevant for mineral weathering, tailings were sampled at five sites up to a depth of about one meter. For microbial community analysis, the molecular methods quantitative real-time PCR (Q-PCR), catalyzed reporter deposition-FISH (CARD-FISH) as well as cloning and subsequent sequencing of 16S rRNA genes were performed besides total cell counting. In addition, the MPN-technique was used to enumerate acidophilic chemolithoautotrophic Fe(II)- and sulfur-oxidizing microorganisms. In parallel, novel acidophilic halotolerant Fe(II)-oxidizing microorganisms were enriched at salt concentrations up to 1 M NaCl. To understand microbe-mineral interaction in this extreme environment, additional (bio)geochemical and mineralogical analysis using microcalorimetry, mineral liberation analysis (MLA) and x-ray fluorescence analysis (XRF) were conducted. Microcalorimetric measurements of the tailings samples revealed that a high proportion of pyrite oxidation is caused by microbial activity of acidophilic Fe(II)- and sulfur-oxidizers. Therefore, metal sulfide oxidation is the main energy source for chemolithoautotrophic microorganisms in the tailings dump at Chañaral Bay. The CARD-FISH analysis of the pyrite-containing tailings detected slightly lower cell numbers than the total cell counts of up to 108 cells g-1 dry weight, which shows that a high proportion of detectable microorganisms is alive. Q-PCR results showed that Bacteria dominated over Archaea. Furthermore, the acidophilic Fe(II)- and/or sulfur-oxidizing Acidithiobacillus spp. were the dominant iron- and sulfur-oxidizing bacteria. The acidophilic Fe(II)-oxidizing Leptospirillum spp. were below detection limit of 103 cells g-1 dry weight. Besides, Sulfobacillus spp., Acidithiobacillus caldus, Geobacteraceae, and sulfate-reducing Bacteria were quantifiable with up to 105 DNA copies g-1 dry weight. Sequencing of the bacterial 16S rRNA gene clone libraries of the tailings samples indicated a frequent occurrence of the phyla Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes. In the enrichments the genera Acidiphilium and Sulfobacillus were found. The geochemical conditions and microbial communities are very different at the different tailings sites and depths which is reflected by a changing microbial community composition.

124A Thermophilic sulfate reducing bacteria from a Brazilian Petroleum Reservoir Elisa Korenblum*1, Camila França2, Monica Penna3, Jan Dirk van Elsas4, Lucy Seldin2 1RUG, UFRJ, Netherlands, 2UFRJ, Brazil, 3CENPES - PETROBRAS, Brazil, 4RUG, Netherlands

Thermophilic sulfate reducing bacteria were isolated from crude oil of a petroleum reservoir, located at Caratinga Field, CamposBasin, Southeast of Brazil. The crude oil recovered from this field had a low water content. The samples were collected from three production wells (CRT21, CRT37 and CRT40) and enrichment cultures were obtained from each production well using Postgate E medium. These cultures were able to grow in a temperature range of 55-80oC, with an optimum at 75oC. A mineral medium containing sulfate (as the electron acceptor) and 1% sterilized crude oil (as the only carbon source) was used to grow these cultures, which were incubated at 75oC for two months, in anaerobic

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conditions. DNA was extracted from the bacterial communities present in both the crude oil samples and the enrichment cultures. The community structures of the enrichment cultures and the crude oil samples were characterized by 16S rRNA gene-based analysis. PCR-DGGE approach indicated only few differences in bacterial composition among the cultures, and also showed that the crude oil samples harbored different bacterial community. However, bands at the same position of bands as in the enrichment cultures were found in the three crude oil samples. Richness estimation based on DGGE bands indicated low diversity in crude oil samples (ca. 7 bands), but were higher than in the enrichment cultures. We have obtained thermophilic sulfate reducing bacteria that grow in crude oil as the only carbon source, which might play a major role in the indigenous bacterial activities in the Caratinga Field.

125A Microbial hydrocarbon degradation to methane in different deep geological systems Martin Krüger*1, Hans-Hermann Richnow2, Heribert Cypionka3, Sabrina Beckmann3 1BGR, Germany, 2UFZ, Germany, 3ICBM, Germany

Since decades it is known from stable isotope studies that large amounts of biogenic methane are formed from higher hydrocarbons in oil, coal and shale reservoirs, but also in “natural systems” like mud volcanoes or deep marine sediments. These geosystems often contain large amounts of organic carbon. In contrast, the availability of alternative electron acceptors usually is limited. Consequently, the degradation of the often very recalcitrant carbon compounds often proceeds under methanogenic conditions. The already low energy gain of these processes has to be shared by syntrophic consortia of several different microorganisms, thus becoming even smaller for the individual partners. While about the degradation of hydrocarbons under sulfate- or nitrate-reducing conditions a lot is known, the enzymatic mechanisms and microorganisms involved in the methanogenic processes have only recently come into focus. The main objective of this work therefore is the biodiversity and metabolic processes involved in the degradation of hydrocarbons to methane. We started to investigate the physiological characteristics and activities of microbial consortia originating from very different habitats. These have been enriched on oils, coals or single hydrocarbons. Isotopic measurements showed the conversion of 13C-labeled substrates into methane. Combining SIP with T-RFLP, Q-PCR and sequencing analysis a large bacterial diversity was detected while the archaeal one was limited to three or four dominant species. Genes indicative of metal reduction, sulphate reduction, and methanogenesis were detected in high numbers in these incubations. The low phylogenetic diversity of Archaea comprised Euryarchaeota and Crenarchaeota. Members of Methanosarcinales and Methanomicrobiales dominated the archaeal part of the community in the microcosms. The main bacterial representatives in the enrichment cultures were Syntrophus spp., Desulfovibrio spp. and Syntrophomonas spp. For the characterization of degradation pathways metabolite spectra are analysed, again combined with the use of SIP. The variability of carbon and hydrogen isotopes of produced methane indicates a common methanogenic degradation mechanism resulting in consistent patterns of hydrocarbon alteration. Mass balance calculations showed that significant fractions (2-10%) of added coals or oils were converted to methane.

125B Osmoadaptation of halophilic methanogen Mei-Chin Lai*, Shu-Jung Lai, Shu-Yao Wu, Chuan-Chuan Hung Hung National Chung Hsing University/Department of Life Sciences, Taiwan

The osmotic strength of the environment is one of the parameters that determine the ability of organisms to proliferate in a given habitat. Changes in the extracellular osmolarity have the same physiochemical effects on cell from all living domains and the responses to osmotic shifts have considerable similarities in all organisms. Strict anaerobic methanogenic archaea can survive at broad range of salt stress (0-4.5 M NaCl) from freshwater, marine to hypersaline environments. The extreme and board range of salt adaptations made the Methanoarchaea the best model for studying salt-stress response of archaea and osmo-adaptation.

To encounter the changing osmotic stress in hypersaline environments, halophilic methanogen- Methanohalophilus portucalensis could transport compatible solute (osmolyte) betaine through an ABC betaine transport system (Bta) which was regulated by betaine and salt stresses. Without the extracellular supply of betaine, cell could de novo synthesize betaine, Nε-acetyl-β-lysine, β-glutamate and β-glutamine as compatible solutes to increase the internal osmolarity and protect cellular macromolecules. N-acetyl-β-lysine was synthesized from lysine through lysine 2,3-aminomutase

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(ablA) to form β-lysine and the acetyl group was further transferred to β-lysine by β-lysine acetyltransferase (ablB). The ablA and ablB genes were screened and obtained through PCR and Southern hybridization techniques from the marine Methanosarcina mazei N2M9705, halotolerant Methanocalculus chunghsingensis K1F9705b and halophilic M. portucalensis FDF1. Northern hybridization results showed that ablA and ablB were co-transcribed and up-regulated while encountering salt up-shock stresses.

Glycine betaine are de novo synthesize from glycine through three steps of methylation, with intermediates sarcosine and dimethylglycine, to form betaine. The complete gene cluster of Mpgsmt-sdmt was obtained and heterologous expressed for in vitro activity assays. The transcription levels of Mpgsmt-sdmt were induced by temperature and salt stresses which were verified by Northern hybridization. Comparison with GSMT from other halophilic bacteria or cyanobacteria, MpGSMT have unique characteristics that dramatically up regulated by potassium and sodium concentrations. The secondary and quaternary structure analyses of MpGSMT showed the dimer formation as increased potassium level that caused the substrate glycine and sarcosine binding affinity improved. The dramatic activating effects of sodium and potassium ions on the glycine and sarcosine methyltransferase activities of MpGSMT, but not MpSDMT and bacterial GSMT/SDMT, and the strong end product betaine inhibitory effect on MpGSMT suggested it is a key player in osmo-regulation. And the result suggested that both salt in and osmolyte strategies are used in osmo-adaptation of halophilic methanogen.

126A Chemolithotrophic activity in a subzero hypersaline spring of the High Arctic Guillaume Lamarche-Gagnon*1, Charles Greer2, Lyle Whyte1 1McGill University, Canada, 2Biotechnology Research Institute, Canada

The Lost Hammer (LH) spring of the Canadian High Arctic is for the only known terrestrial methane seep in a cryoenvironment on Earth present in the form of a hypersaline (24% salinity), subzero (-5°C), perennial spring arising through thick permafrost. The extreme conditions of the LH spring (oxygen depleted, cold temperature, and hypersalinity) make it a prime target for expanding our knowledge on adaptations in cryoenvironments and assessing the limits of terrestrial microbial life. The methane emanating nature of this spring also furthers its importance as an astrobiological analogue considering that methane is seen as a potential biomarker in extraterrestrial studies on frozen worlds such as Mars and Enceladus. Previous studies on LH sediments succeeded in isolating cold-adapted halotolerant heterotrophic bacteria, and provided molecular evidence for the presence of methane oxidizing archaea (ANME). Whether LH microbial communities are active under such hypersaline and cold conditions, however, was not demonstrated. Here, we present preliminary data regarding chemolithotrophic microbial processes hypothesised to occur at LH, as well as biodiversity surveys of deeper, more anaerobic sediment regions of the spring. Whereas anaerobic methane oxidation, and methanogenesis, could not be detected in microcosm incubations using radiolabelled methane (14CH4) and methanogenic-substrate amendments, respectively, hydrogenotrophic sulfate reduction (SR) to sulphide was recorded in samples incubated under laboratory settings that mimicked natural environmental conditions. Sulphide production rates averaged 35 ± 7 nmol•g-1 sediment•day-1 over a span of 200 days at both -5°C and 5°C. 16S rRNA pyrosequencing analyses of DNA and RNA extracted from field-collected samples also indicated that sequences related to ammonia-oxidizing Thaumarchaeota dominated the archaeal datasets, revealing how ammonia oxidation also most likely play important roles in sustaining active microbial lifein situ. Taken together, RNA-based analyses, in concert with sulphide production assays, portray the LH spring as a biologically active environment, hinting at how life can live in such extreme conditions on Earth, and possibly on other planetary bodies bearing similar environmental settings.

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127A Does geochemistry drive community structure and function in high Arctic snow ecosystems? Catherine Larose*1, Emmanuel Prestat2, Sébastien Cecillon1, Aurélien Dommergue3, Timothy M. Vogel1 1Environmental Microbial Genomics, Ecole Centrale de Lyon, Université de Lyon, France, 2Earth Science Division, Ecology Department, Lawrence Berkeley National Laboratory, USA, 3Université Joseph Fourier – Grenoble 1 / CNRS, LGGE, France

Arctic environments are perturbed by a number of stresses, notably those associated with atmospherically transported elements and contaminants, such as heavy metals and organic pollutants. Understanding the effects of these inputs on sensitive ecosystems is critical for gaining insight into global biogeochemical cycles. The microbial community in snow is at the interface of the Arctic ecosystem and the atmosphere and as such provides insights into ecosystem response to these stresses.

An important feature of the Arctic is seasonal snow-cover, which extends over a third of the Earth’s land surface, covering up to 47 million km2, and influences global energy and moisture budgets and thus climate. The snowpack can be considered as a dynamic habitat of limited duration that acts as a medium and a mediator by transmitting and modifying interactions among microorganisms, plants, animals, nutrients, the atmosphere and soil.

In order to uncover the fate of atmospherically derived contaminants in Arctic snowpacks, we explored interactions between microbial communities and their chemical environment in the field using a variety of metagenomic and chemical tools. We linked changes in microbial community structure to snowpack and meltwater chemistry using co-inertia analysis (p=0.006) and explored changes in community function using a pyrosequencing approach. Based on these analyses, nitrogen, sulfur and mercury were shown to influence both community structure and function. In turn, snow microbial communities also have the potential to alter the biogeochemical cycles of these elements, leading to questions regarding the influence of snow microorganisms on ecosystem functioning in the Arctic.

128A 16S rRNA and functional diversity: contrasting fingerprints in arsenic-rich sediments from an acid mine drainage Béatrice Lauga*1, Anne Fahy1, Philippe Bertin2, Denis Le Paslier3, Claudine Médigue4, Jean Weissenbach5, Ludovic Giloteaux1, Robert Duran1 1Université de Pau et des Pays de l'Adour, France, 2Génétique Moléculaire, Génomique et Microbiologie, UMR CNRS 7156, France, 3Commissariat à l'Énergie Atomique et aux Énergies Alternatives, Institut de Génomique, Genoscope, France, 4UEVE, Université d'Évry, CNRS-UMR 8030, France, 5Commissariat à l'Énergie Atomique et aux Énergies Alternatives, UEVE, Université d'Évry, CNRS-UMR 8030, France

The distribution of organisms is a fundamental aspect of ecology and thus of microbial ecology. The Baas Becking's statement has been the basis of increasingly numerous studies made possible by the advent of culture-independent methods, such as ribosomal DNA analysis. While such methods have revealed a vast microbial diversity, much work is still necessary to understand the processes driving patterns of microbial distribution. Because extreme environments place important constraints on species diversity, these are good candidates for the study of microbial ecology and evolution. Acid mine drainage (AMD) is such an extreme environment.

In order to investigate the bacterial diversity and molecular diversity with regards to high levels of arsenic in acid mine drainage (AMD), the metabolically active bacteria of an arsenic-rich AMD sediment from Carnoulès (France) was studied using 16S rRNA cDNA, in parallel to the diversity of functional genes involved in arsenic detoxification (arsB, ACR3) and arsenite oxidation (aioA).

This investigation showed that metabolically active bacteria were affiliated mainly to AMD specialists, that is organisms detected in or isolated from AMDs throughout the world. They included Thiomonas, Acidithiobacillus, Acidobacteria and Actinobacteria spp., as well as unclassified Gammaproteobacteria and non-affiliated organisms close to "Candidatus Fodinabacter communificans" CARN1, a novel organism detected at Carnoulès. The distribution range of these organisms suggested that these show niche conservatism. The deduced protein sequences of arsenite transporters (ArsB, Acr3p) showed

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some diversity, whereas a single type of deduced sequence of arsenite oxidase (AioA) was found. Our data suggested that the aioA gene was more recent at Carnoulès than the genes encoding arsenite transporters, and subjected to a different molecular evolution. In contrast to the 16S rRNA genes already associated with various AMD environments, the functional genes aioA, ACR3, and to a lesser extent arsB, were either novel, or specific to Carnoulès, raising the question as to whether these functional genes are specific to high concentrations of arsenic, AMD-specific, or site-specific.

129A A metagenomic study and active 16S rRNA pyrosequencing analysis of a High Arctic subzero hypersaline spring Chih-Ying Lay*1, Nadia Mykytczuk1, Etienne Yergeau2, Guillaume Lamarche-Gagnon1, Charles Greer2, Lyle Whyte1 1McGill University, Canada, 2Biotechnology Research Institute, National Research Council Canada, Canada

The cold saline springs on Axel Heiberg Island are among the only known cold springs in thick permafrost on Earth and represent unique, subzero environments for expanding our knowledge of microbial life. Lost Hammer (LH) spring is the most extreme and is characterized by perennial subzero temperature (-5°C), hypersalinity (24% salinity), reducing (~165 mV), microaerophilic, oligotrophic conditions, rich in sulfates (100,000 mg/kg), dissolved H2S/sulfides (~ 25 ppm), ammonia (~381 μM), and methane emissions (11.1 g/day). Although the microbial composition in LH spring sediment was determined by 16S clone libraries, the active microorganisms therein, and their functional metabolic capacities remain unknown. In this study, metagenomic analysis of LH source DNA was performed by 454 pyrosequencing and then annotated via the MG-RAST pipeline. Total RNA from the LH source was collected for active microbial profiling, using RNA/cDNA extraction/reverse-transcription techniques followed by 16S rRNA pyrosequencing, and then analyzed via the RDP pyrosequencing pipeline. The objectives of this study were 1) to identify which bacteria and archaea are active in situ, and compare this data with the metabolic pathways characterized; 2) to map the microbial metabolic pathways present and genes associated with environmental stressors; 3) to compare the functional potential of the LH metagenome to metagenomes from other environments, using principle component analysis (PCA) based on the relative abundance of all MG-RAST subsystems. In this study, we found a large proportion of microorganisms identified through the metagenomic approach, were not found to be part of the in situ active microbial community based on 16S rRNA analyses. For example, the most abundant microorganisms from the metagenomic data set (45.07%), Cyanobacteria, were only detected to be a small component (0.09%) of the active microbial community. In the active community, functional groups involved in nitrogen cycle, methane metabolism, sulfate reduction, were detected, that is ammonia oxidizers (Thaumarchaeota), denitrifiers (Pseudomonas spp.) methanotrophs (Verrucomicrobia spp.), sulfate reducers (Desulfobulbus spp.) and other sulfur cycle related species (Thermoprotei). Functional genes involved in these metabolic pathways were also identified in the LH metagenome. By comparing functional community composition to other metagenomes, genes related to sulfur metabolism and dormancy were found to be the major component that distinguished the LH microbial community from the metagenome. This study provides insights into the metabolic potential and active microbial components that exist in this hypersaline high Arctic spring cryoenvironment and will contribute to our understanding of microbial ecology in extreme environments.

130A Microbial diversity and biogeochemical function of the phototrophic microbial mats in epsomitic Hot Lake, WA Steve Lindemann*, James Moran, Young-Mo Kim, Alice Dohnalkova, David Kennedy, Sergey Stolyar, H. Steven Wiley, Allan Konopka, Jim Fredrickson Pacific Northwest National Laboratory, USA

Hot Lake is a meromictic, hypersaline lake that occupies a small glacial endorrheic basin in north-central Washington. It is a dynamic system; besides variability in temperature and photon flux throughout the annum, Hot Lake also experiences a seasonal cycle in salinity, varying between observed extremes of ~200mM MgSO4 after snowmelt to greater than 2M at the close of the dry season. Despite such large environmental fluctuations, an active benthic, phototrophic mat resides in the lake. Cyclic variations in environmental parameters permit analysis of the compositional and functional stability of this community under diverse and athalassohaline chemiosmotic conditions and elucidation of the mechanisms that impart robustness. Observed δ13C ratios for dissolved organic carbon (-19.26‰ ± 0.17‰) are remarkably consistent throughout the lake and are not indicative of terrestrial carbon sources, suggesting the presence of a dominant primary production pathway within

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the lake and making the Hot Lake mat an opportune system to study the biogeochemical impact of changes in community function upon carbon and energy cycling.

This project aims to understand the community structure dynamics and function in response to the annual cycle of the major physicochemical parameters of Hot Lake, especially salinity, temperature, and photon flux. Preliminary studies on the structure of the Hot Lake mat community in July utilized a Sanger-sequenced clone library of rrnA as well as pyrosequencing of this gene. Kmer-based classification using mothur v.1.24.0 revealed evidence of phylotypes consistent with oxygenic and anoxygenic photosynthesis, sulfur oxidation and reduction, and halotolerance. Overall, in July, the mat is dominated by members of Alphaproteobacteria (43.2% of total reads) and, specifically, Rhodobacteraceae (35.5% of total reads). Oxygenic photosynthesis within the mat appeared to be driven by one dominant cyanobacterial operational taxonomic unit (OTU) of order Oscillatoriales (8.9% of reads). Carbonate minerals precipitated beneath the cyanobacterial stratum by an as yet-unknown mechanism. Halophilic purple sulfur bacteria of genus Halochromatium and Thiohalocapsa compose the anoxygenic phototrophic guild, and16S sequences consistent with the presence of bacteria similar to the obligately chemolithotrophic sulfur oxidizer Thialkalivibrio as well as the halophilic deltaproteobacterium Desulfosalsimonas suggest active sulfur cycling within the mat. Pyrosequencing results from samples collected along the depth/salinity gradient in July showed that purple sulfur bacteria are more dominant in mats nearer the lake surface, while elevations in OTUs classified in phylum Spirochaeta and Clostridium in the region of the halocline suggest an increased role for anaerobic degradation of mat biomass in the transition from the oxic epilimnion to the anoxic hypolimnion. Seventeen unique strains have been isolated from the mat to date, including previously uncultivated Maritimibacter, Marinobacterium, and Salinibacterium species. Two filamentous cyanobacteria have been grown in unicyanobacterial simplified natural communities (SNCs) with bicarbonate as the sole carbon source. These cultures are currently being analyzed by GC-MS and stable isotope probing to determine which organic compounds are exchanged between phototrophs and associated heterotrophs. In collaboration with the Joint Genome Institute, metagenomes of these SNCs are being sequenced to generate a predictive model of their metabolism.

131A Bacterial diversity in the foreland of the Tianshan No. 1 and Dongkemadi Glacier, China Guangxiu Liu, Wei Zhang*, Xiukun Wu Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, China

There is compelling evidence that glaciers are retreating in many mountainous areas of the world due to global warming. With this glacier retreat, new habitats are being exposed that are colonised by microorganisms whose diversity and function are less well studied. During our recently researches, we characterised bacterial diversity along the chronosequences of the Glacier No. 1 and Dongkemadi glacier foreland that follows glacier retreat in China, by using culture-dependent and 454 pyrosequencing method. Results showed that the culturable bacteria numbers increased with glacier terminus retreat distance, and significantly positively correlated to the soil total N and C and the soil water content, as well as the soil urease, sucrase, dehydrogenase, catalase, and polyphenol oxidase activities. Such results suggesting that the soil bacteria were involved in the procedure of soil formation and improvement in glacier foreland. Based on 16S RNA gene identification, the predominant cultivable bacteria were Actinobacteria , Bacteroidetes and Proteobacteria. An average of 10,000 sequences was obtained from each sample by 454 pyrosequencing. Using non-parametric and rarefaction estimated analysis, we found bacterial phylotype richness was high and increased with glacier terminus retreat distance. Pyrosequencing showed tremendous bacterial diversity, among which the Acidobacteria, Actinobacteria, Bacteroidetes and Proteobacteria were found to be present at larger numbers at both glacier foreland. Meanwhile, the proportion of Bacteroidetes and Proteobacteria decreased and the proportion of Acidobacteria and increased along the chronosequences. These findings suggest that high-throughput pyrosequencing can comprehensively detect bacteria in the foreland, including rare groups, and give a deeper understanding of the bacterial community structure and variation along the chronosequences. These results provide the database for the bacterial diversity information and bacterial succession rules at glacier foreland.

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132A Cold-active and alkali-stable proteases from bacteria isolated from the polyextreme ikaite columns in Greenland Jeanette Eva Lylloff*, Mikkel Glaring, Peter Stougaard Copenhagen University, Denmark

The ikaite columns found in Ikka Fjord in South Western Greenland constitute a very rare environment. The interior of the columns is cold (4°C), alkaline (pH 10.4), and display low salinity (0.9%) and harbor a microbial community adapted to this polyextreme environment.

The aim of this study is to identify novel cold-active proteolytic enzymes from cold-adapted bacteria from the ikaite columns.

Cultured bacteria were isolated directly from ikaite material on different types of media and screened for production of proteolytic enzymes. The genomes of two bacterial isolates were sequenced and genes predicted to code for proteolytic enzymes were identified by bioinformatics. Currently, selected protease-encoding genes are being cloned and recombinantly expressed. Proteolytic enzymes will be characterised in relation to industrial applications, e.g. substrate specificity and pH-, salt-, and temperature-stability and -optimum.

Application of the cold-active proteases may lead to low temperature processes, for example laundry processes, hydrolysis of food proteins, and production of bioactive peptides.

133A Unique genomic and physiological traits of an ultra low-diversity bacterial and fungal high-elevation (> 6000 m) cold desert mineral soil community Ryan Lynch*, Steve Schmidt University of Colorado Boulder, United States

High-elevation, plant and glacier free soils rank among the most physiochemically extreme and oligotrophic terrestrial environments. The mountain topography and low atmospheric pressure drive daily temperature fluctuations across the freezing point (with an amplitude of up to 70°C), and generate periodic snowfall. Building upon our initial microbial community and biogeochemical assays of the mineral soils of Volcán Llullaillaco (6739 m elevation) and Volcán Socompa (6051 m elevation), which are stratovolcanos that rise above the hyperarid Atacama desert, we now report our initial results from both deep-coverage metagenomic sequencing efforts and related physiological experiments.

We deployed the long-read 454 GS FLX+ sequencing system (mode read length > 600 bp) to randomly fragmented bulk nucleic acid extractions from high-elevation, low-biomass soil samples. We utilized a modified serial silica filter binding protocol to overcome low DNA yields, which allowed us to avoid the potential biases introduced from random genomic amplification techniques. Sequence binning and classifications were performed using the MG-RAST and PhymmBL approaches. Sequence contig assembly, comparative genomic recruitment, and genetic analyses were performed using the Geneious suite. Microbial isolations and growth tests were carried out under a variety of temperature and water availability regimes that were modeled from field site data.

Our results confirm that like many arid desert, polar, and alpine settings, Actinobacteria dominate both bacterial and overall microbial abundance. Specifically, soils from 6030 m elevation on Volcán Llullaillaco are dominated by a highly divergent spore-forming filamentous Pseudonocardia sp. Kilobase scale high coverage contigs reveal extensive levels of horizontal recombination from both other Actinobacteria, as well as other bacterial phyla. At the community level we also reconfirm the absence of any complete photosynthetic pathways, yet find an abundance of genes that code for the both the complete Calvin-Benson and C4-dicarboxylic acid CO2 fixation cycles. The presence of complete gene sets for the aerobic oxidation of carbon monoxide, hydrogen, methane, and nitrate suggest these atmospheric substrates may provide energy for chemoautotrophy in this system. Overall, we hypothesize that the majority of biomass resides below the top centimeters of soil that light penetrates. Here microbial life finds a limited buffer from the hyperarid and solar scorched surface, and relies on trace gases, aerosols and other Aeolian inputs to survive during periodic episodes of sufficient snowmelt derived water availability. Our freeze-thaw cycle isolation and growth experiments provide further evidence for the selectivity of this environment, and suggests how the resident

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microbes have adapted to this niche. All isolates are psychrotolerant yeasts and bacteria capable of strong growth under a daily freeze (-4°C) thaw (40°C) cycle that mimics temperature data collected at 4 cm soil depth from 6000 m elevation.

134A The ecological dichotomy of ammonia oxidizing archaea and bacteria in the hyper-arid soils of the Antarctic Dry Valleys Catarina Magalhães*1, Adriano Bordalo2, Ana Machado2, Béatrice Barbier3, Charles Lee3, Craig Cary3 1CIIMAR, Portugal, 2ICBAS, University of Porto, Portugal, 3International Centre for Terrestrial Antarctic Research, University of Waikato, New Zealand

The McMurdo Dry Valleys of Victoria Land is the largest ice-free areas Antarctica and considered to be the most physically and chemically extreme terrestrial environment on Earth. In such harsh conditions, microorganisms dominate and are believed to drive key biogeochemical processes in the system; however fundamental questions on the biodiversity and environmental controls of these microbial communities remain unanswered. In this study we investigated the diversity and abundances of archaeal (AOA) and bacterial (AOB) ammonia oxidizers in four Dry Valleys with highly variable soil geochemical properties: Miers Valley (MV), Upper Wright Valley (UW), Beacon Valley (BV) and Battleship Promontory (BP). Results revealed clear differences in soil bacteria 16S rRNA gene copy numbers, with one order of magnitude more bacterial biomass in MV and BP compared with the BV and UW Valleys. In agreement with the quantitative data, pyrosequencing of 16S rRNA gene and ARISA analyses showed significantly higher levels of bacterial diversity in MV and BP compared with BV and UW. Generally low AOB and AOA amoA genes diversity was observed; from a total of 210 clones of AOA and AOB amoA, only four AOA and AOB OTUs were recovered in the four Dry Valleys. However, clear differences were observed in the relative abundances of AOA and AOB among the Dry Valleys evaluated. While beta-proteobacterial AOB amoA genes dominated the ammonia-oxidizing community in soils from MV and BP, higher abundance of AOA amoA genes was found in UW and BV soils. In agreement, pyrosequencing analysis recovered AOB 16S rRNA sequences only at MV and BP valleys. Our results suggested that the extremely harsh conditions of UW and BV soils characterized by colder year-round temperatures, lower availability of liquid water and much higher conductivity values may connected with the lower observed bacterial diversity and abundance, and the numerical dominance of AOA within the ammonia oxidizers microbial communities of these extreme Antarctica ecosystems.

135A Metagenomic analysis of microbial community inhabiting the deep subsurface thermal waters in Western Siberia Andrey Mardanov*, Vitaly Kadnikov, Nikolay Ravin Centre Bioengineering RAS, Russian Federation

The deep subsurface ecosystems are characterized by anaerobic conditions, extreme pressure and high temperature. We analyzed the microbial community of the underground thermal waters springing out of a 2775 meters deep abandoned oil-exploration borehole in Tomsk region, Western Siberia, Russia. The borehole was drilled into putatively Mesozoic sedimentary rocks of the Western Siberia basin about fifty years ago but no oil was found. The outcoming water is anoxic, neutral-to-slightly alkaline with a temperature of 46-51°C. Two approaches were used to characterize the microbial community: identification of microorganisms based on amplification and pyrosequencing of variable V3-V5 fragments of 16S rRNA genes, and sequencing of the total community DNA. The 16S rRNA analysis indicated the dominance of bacteria in the clone library (81% of reads), while hydrogenotrophic methanogenic archaea of the genus Methanothermobacter accounted for the rest. The most abundant lineage, Firmicutes (76% of all reads), was represented by sulfate-reducing bacteria of the genera Desulfovirgula (54%) and Desulfotomaculum (10%), as well as by organisms related to Thermacetogenium (7%), Pelotomaculum (0.2%), and Moorella (0.3%). The rest of bacterial 16S rRNA sequences was assigned to OP9 phylum (3%), gamma-proteobacteria (0.1%), Bacteroidetes / Chlorobi group (0.2%; half of these clones belongs to recently described phylum Ignavibacteriae), and uncultured lineages.

Metagenomic DNA sequencing revealed the presence of four dominant species in the community, - Desulfovirgula thermocuniculi, Desulfotomaculum kuznetsovii, Thermacetogenium sp. and an unknown Firmicutes bacterium. Contrary to the results of 16S RNA analysis, methanogenic archaea

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accounted for only few percents of metagenome indicating that archaea were overrepresented in the 16S rRNA library. The results of metagenomic analysis suggest the following ecological relationships between microorganisms. Desulfovirgula thermocuniculi and Desulfotomaculum kuznetsovii are sulfate-reducing bacteria that can anaerobically oxidize low-molecular organic compounds and intermediate metabolites (lactate, butyrate, propionate) to acetate, and then to carbon dioxide. Bacteria of genus Thermacetogenium can hydrolyze organic polymeric compounds. Other non-sulfate-reducing Firmicutes, and possibly the organisms belonging to Ignavibacteriae can also hydrolyze such compounds. Overall the results of this study provide new information regarding previously uncharacterized environment and show the value of high-throughput sequencing in the study of complex ecosystems.

136A Diversity of culturable anaerobic bacteria in rhizosphere soil from Antarctic vascular plant of Admiralty Bay, maritime Antarctica Rafael José Marques Peixoto*1, Leandro A. Lobo2, Karla Miranda2, Geraldo Renato Paula3, Naiara Rust2, Alexandre Rosado2, Raquel Peixoto2, Regina Maria Domingues2 1Universidade Federal do Rio de Janeiro, Instituto de Microbiologia Paulo de Góes, Brazil, 2UFRJ, Brazil, 3UFF, Brazil

Due to the low temperatures and its geographic isolation, the Antarctic continent presents singular territory characteristics, both in terms of climate and composition of ecosystems. This unique ecosystem has the energy balance of biogeochemical cycle and food chain largely influenced by the activities of microorganisms. The microorganisms found in this region hold valuable information concerning the evolutionary history of our planet, in particular anaerobic bacteria, which are regarded the first prokaryotes to evolve on earth. A 16S rRNA pyrosequencing studies have revealed an intriguing predominance of anaerobic bacteria in the rhizosphere of vascular plants in King George Island, Antarctic. Despite the difficulties inherent in anaerobic bacteriology, this work is based on the isolation of anaerobic bacteria presents in the rhizosphere of vascular plant Deschampsia antarctica, and characterization creating a growth profile of metabolites and enzyme expression, and their possible biotechnological applications. The samples were collected and transported in anaerobic conditions in the summer of 2010/2011. The specimens were either directly processed or heat-shocked for 30 min at 60°C and the isolation was performed using spread-plate (anaerobic incubation) or oxygen gradient (in Thioglycolate) techniques with several parameters, which included: NaCl concentration (0,5%, 1,5% e 2,5%), pH (6, 7 e 8) and temperature (5°C, 15°C, 25°C e 50°C) attempting to recover a greater amplitude of anaerobic organisms. Initially 172 isolated bacterial were obtained, 57 of which were strict anaerobes. Subsequently, the complete 16S ribosomal gene of these isolates was amplified and sequenced. The results indicate the prevalence of the genus Clostridium, albeit the isolation of a large number of previously unidentified species. In general we observed a predominance of the phylum Firmicutes and Actinobacteria among the isolates, which is in agreement with previous works.

137A Identification of genes involved in zinc and cadmium adaptation in rhizobia isolated from heavy metal mine soil Geraldine Maynaud*1, Antoine Le-Quere2, Erika Yashiro3, Jean-Claude Cleyet-Marel1, Brigitte Brunel1 1Laboratoire des Symbioses Tropicales et Méditerranéennes, France, 2IRD, LMI Rabat, Morocco, 3Department of Plant Pathology, University of Wisconsin - Madison, WI, USA

An efficient nitrogen fixing symbiosis between a legume, Anthyllis vulneraria, and its rhizobial partner, Mesorhizobium metallidurans, was identified in a highly polluted mining site in the South of France. Soil samples extracted from this mining site were shown to be highly polluted with heavy metals (EDTA-extractable: 2.600 mg kg-1 Zn, 7.430 mg kg-1, Pb, 19.5 mg kg-1 Cd). This Anthyllis-Mesorhizobium symbiotic pair has recently been described as a key potential bioremediation agent and pivotal to stimulate a new plant cover which could then limit the movement of the heavy metals from the contaminated sites>. M. metallidurans strains have also been shown to be resistant to high Zn and Cd concentrations (16-32 mM of Zn and 0.3-0.5 mM of Cd).

The aim of our work is to identify and characterize genes involved in heavy metal adaptation in M. metallidurans.

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Two complementary approaches have been developed, (i) a genetic-based approach consisting of functional screening of a cosmid library of M. metallidurans STM2683T on Zn- or Cd- supplemented medium, and subsequent sub-cloning and sequencing of DNA loci exhibiting heavy metal resistance and (ii) a global approach based on the transcriptome sequencing (RNAseq) corresponding to Zn, Cd or no metal treatments and comparative analyses of two Mesorhizobium isolates (M. metallidurans STM2683T and another metal resistant Mesorhizobium sp. strain).

The 2 approaches have allowed the identification of a gene (named mmcadAI), coding a P-type ATPase, specifically induced by Zn and Cd, and homologous to CadA of Staphylococcus aureus that is involved in Cd, Zn and Pb export. Phenotypic analysis revealed that mmcadAI contributes to Zn and Cd resistance but not to Pb. The mmcadAI-deleted mutant in STM 2683T was affected at high Zn concentrations compared to the wild-type strain. However, the mmcadAI deletion did not completely abolish the resistance down to a level comparable to that of a metal sensitive Mesorhizobium strain, suggesting that the heavy metal related adaptation is not due to a single transport system but rather to several interacting systems. Indeed, the RNAseq data revealed that about 50 genes were differentially regulated upon metal treatment, 20 of these genes being homologous between the 2 strains of Mesorhizobium. Among regulated genes, an ABC-type transport system, a ZntA P-type ATPase, a high Zn affinity efflux ZitB, a signal peptidase II that is involved in Pb resistance into Cupriavidus metallidurans CH34 as well as several genes encoding putative proteins whose expression can be associated to heavy metal exposure, were identified.

In conclusion, the 2 complementary approaches identified a novel molecular marker for heavy metal resistance (mmcadAI), involved in the export of Zn and Cd and conferring a high level of resistance to M. metallidurans. The transcriptomics approach also identified other putative genes involved in heavy metal adaptation and which will require further studies to improve our understanding of bacterial adaptation to metal pollution.

138A The proteomic response of extreme acidophile, Acidithiobacillus ferrooxidans, to increased chloride concentrations Timothy McCredden*, Robert Steuart, Christopher Bryan, Elizabeth Watkin Curtin University, Australia

High concentrations of chloride ions inhibit the growth of acidophilic iron and sulfur oxidizing microorganisms used in biomining, a problem particularly relevant to Western Australian and Chilean biomining operations. This growth inhibition is due to the chloride ion disrupting homeostatic mechanisms resulting in acidification of the cytoplasm. Acidithiobacillus ferrooxidans is a found widely in biomining systems however, little is known about the mechanisms this microorganism adopts in order to tolerate sub-lethal chloride ion concentrations. This study applied proteomics to elucidate how A. ferrooxidans alters its proteome under increased chloride concentrations. A. ferrooxidans (DSM 14882T) was grown in the presence of low (0 g.L-1) and high (8 g.L-1) NaCl concentrations, chosen for optimal and sub-optimal growth rates. Total soluble proteins produced by cells were compared using 2D- polyacrylamide gel electrophoresis. Protein spots that were identified as having statistically different abundance between high and low salt conditions were excised, trypsin digested and analyzed by tandem mass spectrometry. Mascot sequence matching software was used to identify proteins of interest. Analysis of differential expression showed that A. ferrooxidans adopted several changes in its proteome in response to NaCl stress. The majority of the strategies represented a general stress response mechanism similar to those induced during pH stress. These included major stress related periplasmic chaperones, whose action is essential for outer membrane formation, and proteins involved in central carbon metabolism, energy acquisition, amino acid synthesis and protein fate. A response that was unique to the NaCl stress response was the accumulation of particular amino acids which have osmoprotectant characteristics. Gaining an understanding of the range of mechanisms that acidophilic iron oxidizing microorganisms may use to help the cell function in the presence of elevated concentrations of chloride can be applied to the development of saline biomining operations or improve alterative processes.

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079B Hot and Cold: Microbial ecology of geothermal soils of Antarctica Ian McDonald*, Craig Herbold, Chelsea Vickers, Craig Cary University of Waikato, New Zealand

Geothermal soils located near the summit of Mt. Erebus, Mt. Melbourne, and Mt. Rittman Antarctica, are some of the most geographically isolated geothermal environments on Earth. We had previously revealed a unique microbial community at Tramway Ridge, Mt. Erebus, that is likely of subsurface origin. In this study we have conducted 16S rRNA based PCR-amplicon pyrosequencing in order to study the microbial community structures in more detail. Pyrosequencing of Tramway Ridge soils unveiled a rich, vertically stratified subsurface ecosystem. The shallow subsurface is dominated by thermophilic nitrogen-fixing Cyanobacteria, Meiothermus, Acidobacteria, Planctomycetes and Bacteroidetes, common constituents of microbial mats at other studied geothermal sites. Their presence in high numbers suggests that the shallow subsurface contains microbes that have seeped from an overlying surface microbial mat composed of cosmopolitan species. The deeper subsurface community is markedly different however and is dominated by a unique archaeon and an OP10-like group that was also observed in our bacterial clone library. Additional members of subsurface community are heterotrophic Chloroflexi and taxa with unknown phylogenetic affilitations that are most similar to deep-branching environmental clones obtained from other subsurface geothermal sites. These findings support our initial hypothesis that the microflora at Tramway ridge is likely subsurface in origin but also suggests that the evolution of this ecosystem has been influenced by a more recent colonization by cosmopolitan microbial mat species. Pyrosequencing has also been used to compare the microbial diversity of the geothermal soils of Mt. Melbourne and Mt. Rittman with those of Mt. Erebus.

139A Population genetic structure of deep-sea chemolithoautotrophs inferred from multilocus sequence analysis Sayaka Mino*1, Satoshi Nakagawa1, Tomoo Sawabe1, Junichi Miyazaki2, Hiroko Makita2, Takuro Nunoura2, Masahiro Yamamoto2, Ken Takai2 1Hokkaido University, Japan, 2JAMSTEC, Japan

Deep-sea hydrothermal fields are areas on the seafloor of high biological productivity fueled primarily by microbial chemosynthesis. Chemolithoautotrophic Epsilonproteobacteria with an ability to utilize inorganic substrates such as H2S and H2 are dominant in deep-sea hydrothermal vents around the world. Beside the non-pathogenic deep-sea chemolithoautotrophs, the class Epsilonproteobacteria contains important human pathogens, i.e. Helicobacter pylori and Campylobacter jejuni. These pathogenic Epsilonproteobacteria have extremely high frequencies of genetic mutation and horizontal gene transfer. Little is known, however, about deep-sea epsilonproteobacterial population genetic structure. In our previous study, we clarified that Epsilonproteobacteria Group B population were geographically separated, and that they had high mutation rates. However, there are still many questions to resolve, e.g. whether these trends are common to all epsilonproteobacterial subgroups and non-epsilonproteobacterial chemolithoautotrophs. In this study, we performed multi-locus sequence analysis (MLSA) on deep-sea vent chemolithoautotrophs of Epsilonproteobacteria Group A, B, F and the genus Persephonella to clarify their population genetic structures.

Chemolithoautotrophic strains used in this study were isolated from chimney structures, vent fluids, and hydrothermal sediments. The hydrothermal samples were collected from geographically separated hydrothermal areas of the South Mariana Trough, Okinawa Trough and Central Indian ridge. We carried out various population genetic analyses including the construction of phylogenetic trees, estimation of mutation and recombination rates based on sequences of various housekeeping genes. In addition, using whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method, we assessed phenotype-genotype relationships.

The MLSA revealed that both deep-sea Epsilonproteobacteria and Persephonella populations were influenced by geographic locations. These deep-sea chemolithoautotrophs appeared to diverge by mutation rather than recombination.

We will discuss the geographical dispersion patterns of deep-sea chemolithoautotrophs and relationship between their genetic and phenotypic traits.

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140A Hydrothermal fluid flow affects the dispersal of subsurface Zetaproteobacteria Craig Moyer*, Sean McAllister Western Washington University, United States

Communities of Fe-oxidizing Bacteria (FeOB) are common at sites of hydrothermal venting and are known to form complex communities in microbial mats, hydrothermal sediments, and borehole fluids. Studies at sites around the Pacific Ocean have found the Zetaproteobacteria to be dominant members of many of these FeOB communities. Preliminary culture-independent analyses indicate that borehole fluids from the deep subsurface may support several novel and distinct Zetaproteobacteria phylotypes (McAllister et al., 2011). Sampling from the deep subsurface at the Iheya North hydrothermal field has provided an opportunity to explore the microbial biodiversity of this unique subsurface habitat.

Onboard enrichment experiments for FeOB were carried out at several sites over a wide range of depths as part of IODP Expedition 331. Enrichment experiments were carried out in both microaerophilic and anaerobic environments, with oxygen and nitrate as terminal electron acceptors, respectively. Although tested for the presence of FeOB from 0.23 to 42.45 meters below the seafloor (mbsf), Site C0014 showed little growth except for in the top half meter of hydrothermally unaltered, weakly oxidized sediment. Site C0015, which was diffusely venting and had a relatively low-temperature at depth (~10.5°C), showed growth throughout the hole with both microaerophilic and anaerobic conditions to a depth of 9 mbsf. Growth at this low-temperature site is consistent with previous studies of iron-oxidizing communities, and may correlate best with the borehole community at the Southern Mariana Trough (Kato et al., 2009). At Site C0017, a potential zone of recharge flow (down-welling of cold oxygenated seawater) was traversed from ~26 to 35 mbsf, and at the top of this zone the microaerophilic enrichment experiments showed growth. Growth in this recharge zone represents the deepest subsurface cultivation of FeOB to date, and may lend insight into the colonization of low-temperature vent systems via subsurface fluid flow.

Molecular analyses of the enrichment experiments and original core samples are currently ongoing. Quantitative PCR (Q-PCR) assays were conducted on original core samples to determine the proportion of Zetaproteobacteria in the total bacterial community, targeting those samples that yielded growth in the enrichment experiments. Most of the samples tested had a low-level detection of Zetaproteobacteria (average 3.65%), with three of the samples having over 12% Zetaproteobacteria in the bacterial community. Further traditional and metagenomic analyses of these samples will allow us to assess the physiology and phylogenetics of these deep subsurface Zetaproteobacteria, as well as the structure of deep subsurface FeOB communities.

141A Genomic and glycomic analysis of endosymbionts and their gastropod hosts in a deep-sea hydrothermal field, Central Indian Ridge Satoshi Nakagawa*1, Shigeru Shimamura2, Tamaki Watanabe1, Tomoo Sawabe1, Ken Takai2, Yoshihiro Takaki2 1Hokkaido University, Japan, 2JAMSTEC, Japan

Deep-sea vents are the light-independent, highly productive ecosystems driven primarily by chemoautotrophs. Most of the invertebrates thrive there through their relationship with symbiotic chemoautotrophs. Chemoautotrophs are microorganisms that are able to fix inorganic carbon using a chemical energy obtained through the oxidation of reduced compounds. Following the discovery of deep-sea vent ecosystems in 1977, there has been an increasing knowledge that deep-sea vent chemoautotrophs display remarkable physiological and phylogenetic diversity. Cultivation-dependent and -independent studies have led to an emerging view that the majority of deep-sea vent chemoautotrophs have the ability to derive energy from a variety of redox couples other than the conventional sulfur-oxygen couple, and fix inorganic carbon via the reductive tricarboxylic acid cycle.

Recent genome studies of heritable endosymbiotic bacteria of insect have provided new insights into the origin and evolution of symbionts and their diverse biological roles in hosts. Similarly, genomic, metagenomic and postgenomic studies have considerably accelerated the comprehensive understanding of molecular mechanisms of deep-sea vent chemoautotrophy, even in unculturable endosymbionts of vent fauna. For example, genomic analysis suggested that there are previously unrecognized evolutionary links between deep-sea vent chemoautotrophs and important human/animal pathogens. However, relatively little is known about the genomic feature of horizontally

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transmitted endosymbionts. In this study, we sequenced the whole genome of the probably horizontally transmitted endosymbionts of gastropod from a deep-sea hydrothermal field in the Central Indian Ridge, as an effort to address 1) genome evolution of horizontally transmitted, facultative endosymbiont, 2) their genomic variability, and 3) genetic differences among symbionts of various deep-sea vent invertebrates. In addition, to assess their response to environmental fluctuation, we determined the host serum N-linked glycan profiles for individuals incubated under various conditions onboard.

The endosymbiont genome was slightly reduced in size, but no AT bias or accelerated genome evolution was apparent, suggesting relatively modern symbiotic relationship. The genome encodes for multiple systems for chemoautotrophic respiration, probably reflecting their adaptation to their niches with continuous changes in environmental conditions. In addition, the endosymbionts appeared to have flagellar, chemotaxis genes, and even sugar PTS systems, suggesting they have the ability to grow heterotrophically in free-living state. Furthermore, we found they had extremely low allelic diversity within the population, reflecting enhanced genetic drift in the small population.

142A Microbiology, geochemistry, and mineralogy of a household sand filter used to remove arsenic from drinking water in Vietnam Katja Nitzsche1, Ankita Bhansali*1, Vi Mai Lan2, Pham Thi Kim Trang2, Pham Hung Viet2, Michael Berg3, Andreas Kappler1, Sebastian Behrens1 1University of Tuebingen, Germany, 2Hanoi University, Viet Nam, 3Eawag, Switzerland

Worldwide more than 100 million people ingest detrimental concentrations of arsenic by consuming groundwater contaminated from natural geogenic sources. Many Asian countries, in particular Vietnam, Bangladesh, India, and Cambodia are known to be affected by high groundwater arsenic concentrations as a result of chemically reducing aquifer conditions. Household sand filters are simple to operate and remove on average 80% of arsenic from groundwater containing 1 mg/L of ferrous iron or an iron/arsenic ratio of about 50. The installation and operation costs of household sand filters are low and the construction materials are locally available. The filters can treat a reasonable amount of groundwater within a short time and they can easily be installed by the affected communities. Oxidation of dissolved iron present in the groundwater leads to the formation of sparsely soluble iron(hydr)oxide particles in the sand filters, which bind negatively charged arsenic species and reduce arsenic concentrations in the water.

Although household sand filters have been proven to be an effective technical solution for mitigating arsenic exposure, not much is known about microbial iron, manganese, arsenic redox processes occurring in the filters and their effect on filter efficiency. Therefore, a goal of this study is to isolate, identify, and quantify Fe, Mn, and As-oxidizing and –reducing microorganisms from a arsenic removal sand filter and to study their specific Fe, Mn, and As redox activities. Water samples and filter solids were collected from a local sand filter close to the city of Hanoi. The samples were geochemically and mineralogically characterized. Total iron, arsenic, manganese, and phosphate concentrations, pH, total organic carbon (TOC), total inorganic carbon (TIC) measurements, as well as total cell counts were performed on samples from various depth of the sand filter. Most probable number counts confirmed the presence and activity of various iron, manganese, arsenic redox-processes and revealed their distribution within the sand filter. Cultivation-based approaches were complemented by 454 sequencing of 16S rRNA gene amplicons for in-depth characterization of the microbial community composition of the sand filter. The findings of this research project contribute to a better understanding of the microbial redox transformation processes that drive arsenic/manganese/iron mineral interactions in arsenic drinking water filters. Knowledge on the biotic and abiotic mechanisms of arsenic (im)mobilization on/from the drinking water filter will enable recommendations for riskless filter use and safe filter material disposal.

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143A Isolation, identification and applications of halotolerant bacteria from São Paulo Zoo Composting Debora Noma Okamoto*1, Lilian Caroline Gonçalves Oliveira1, Patrícia Locosque Ramos1, Márcia Yuri Kondo1, Alyne Marem Silva Barbosa1, Thiago Carlos Bertolin1, Diego Magno Assis1, Rafael Costa Santos Rocha1, João Batista Cruz2, Suzan Pantaroto Vasconcellos1, Luiz Juliano1 1Universidade Federal de São Paulo, Brazil, 2Fundação Parque Zoologico de São Paulo, Brazil

In recent years, investigations to search for biocatalysts that can cope with the conditions of industrial process have been increased. Extracellular degrading enzymes, hydrolases, such as amylases, proteases and lipases are important for microorganisms to utilize organic compounds in different ecosystems and have been used in various fields of industry. Halophiles are an excellent source of such enzymes which are not only salt tolerant, but also may be active at high temperature and pH values. In addition other biotechnological products that have been studied in halophilic/halotolerants microorganisms are Exopolysaccharides (EPS) and Polyhydroxyalkanaotes (PHA). The biosynthesis of these is one of the most common protective mechanisms by extreme environment. In this study we describe the screening of halophilic bacteria for hydrolysis reactions and biopolymers producing. These strains were, isolated from Zoo Park Foundation of São Paulo (ZPFSP) composting process. It was found 8 halotolerant isolates from the composting process of organic residues generated at FPZSP. Based on sequence analysis of 16S rRNA gene these bacteria were identified as Bacillus sp. (SR7), Brevibacterium sp. (YPC12) and Staphylococcus sp. (SR6, SR12, YPC6, YPC8, YPC13, YPC15) genus. According to Kushner classification, seven from the eight isolated microorganisms could be related to halotolerant or extremely halotolerant strains and could grow in the absence of salt as well as in the presence of relatively high salt concentrations (up to 2.5 M). The strain YPC15 (Staphylococcus sp.), could be considered as a halophilic strain and showed optimum growth when cultured in halophilic specific medium added by NaCl 2.5M. The hydrolytic screenings showed that the strains SR6, SR12, YPC8, YPC15 (Staphylococcus sp.), SR7 (Bacillus sp.) and YPC12 (Brevibacterium sp.) it was possible to detect lipolytic, amylolytic or lipolytic and proteolytic activities, which was in accordance to moderately halophilic bacteria abilities. The strain YPC12 was presented as a potential producer of biopolymer (PHA) when octanoic acid and xylose were applied as carbon sources. About the production of EPS with bioemulsifier ability, the isolates were cultured in minimal mineral medium using glycerol as sole carbon source. After 5 days of culture, it was measured the emulsification potential of the produces EPS by the strains. The strains SR6, YPC13, YPC15 and YPC12 showed emulsification indexes more than 55%, when hexadecane was applied as hydrophobic compound model. In conclusion, hydrolyses from halophiles/halotolerants will be of invaluable help for biotechnological applications and the possibility of biopolymers degrading enzymes offer a new solution to the treatment waste where high salinity are typically found.

144A Identification of extracellular proteases in halophiles isolated from La Pampa solar ponds Lilian Caroline Gonçalves Oliveira*1, Roberto Alejandro Paggi2, Debora Noma Okamoto3, Debora Nercessian2, Leonardo Di Meglio2, Maria Aparecida Juliano3, Rosana Esther De Castro2, Luiz Juliano3 1UNIFESP/Infar, Brazil, 2Universidade Nacional de Mar del Plata, Argentina, 3Universidade Federal de São Paulo, Brazil

Halophiles are microorganisms adapted to survive in hypersaline environments. They are an excellent source of enzymes which are not only require high-salt concentrations (1-4 M range) for activity and stability, but also may be active at high temperature and pH values. These adaptations make halophilic enzymes a valuable resource of molecules, including enzymes with potential applicability in Biotechnology. Haloarcula japonica, Haloarcula argentiniensis, Halobacterium sp., Halobacterium salinarum and Salicola sp were previously isolated from the solar ponds (NaCl> 30%, pH 7-8) Guatraché, Colorada Grande and Salitral Negro from La Pampa (Argentina) and identified by means of 16S rRNA analysis. Two different assays were performed to detect extracellular protease activity: a) degradation on solid halophilic medium of casein or hemoglobin; b) detection of protease activity using a randomized library of fluorescence resonance energy transfer peptide (FRET) by cell-free supernatants (Oliveira et al., Analytical Biochemistry 421:299, 2012). These are synthetic combinatorial libraries (SCL) with the general structure Abz-GXXXXXQ-EDDnp where Abz (ortho-aminobenzoic acid), the fluorescence donorlocated at the C-terminus, and Q-EDDnp (glutamine-[N-

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(2,4-dinitrophenyl)-ethylenediamine]) the fluorescence acceptor locatedat N-terminus and X consists of an equimolar mixture of all amino acids. In the sub-libraries Abz-GXXZXXQ-EDDnp the Z position is occupied with a specific amino acid which is absent in the other X positions.On agar plates the proteolytic activity was detected with Haloarcula and Halobacterium genera. These archaea were the only ones that showed activity in the assays performedwith cell-free media and the SCL (Z = X, F, R and V) at pH 8 and 3 M NaCl. The activity in Halobacterium medium showed better activity in a pH range from6.0 to 11.0 for H salinarum and pH 7.0 to 10.0 for Halobacterium sp. When different Z position was used at pH 8 and 3M NaCl, the hydrolytic activities present in the media of both archaea showed similar amino acid preferences in P1 position (Z = V, L, I, F, S, E). The hydrolytic activity was completely abolished in the presence of PMSF (serine protease inhibitor) at pH 6 to 8 while the addition of the ortho-phenanthroline (metallo-protease inhibitor) decrease 50% the hydrolytic activity in both microorganisms. Altogether, these results point to two different enzymes, a serine protease with an optimal pH around 8 and a metallo-protease with optimum activity at pH 10.0.

144B Characterization of tolerance to Arsenic of Exiguobacterium sp. isolated from high altitude Andean wetlands Omar Ordoñez1, Carolina Belfiore1, Daniel Kurth1, Nestor Cortez2, Maria Eugenia Farias*1 1PROIMI-CONICET, Argentina, 2IBR-CONICET, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Argentina

The North West of Argentina is particularly rich in wetlands located in the Puna and Andean region at altitudes in the range of 3,600 to 4,600 m above sea level. Most of these wetlands are completely isolated and are submitted to extreme conditions such as high salinity, wide range of temperatures, high ultraviolet solar B radiation (UV-B), and high content of toxic elements particularly Arsenic. Two strains of Exiguobacterium sp. (named S17 and N30) isolated from Laguna Negra and Laguna Socompa located in Northwestern Argentina showed resistance to high concentrations of Arsenic.

The aim of this study was to elucidate the genetic mechanisms by which Exiguobacterium sp. S17 and N30 resist high arsenic concentrations.

The effect of growth in presence of Arsenic in its two oxidation states (V and III) was evaluated by different methods. Bacterial cultures were subjected to serial dilutions and aliquots of 10 µl were plated onto LB agar supplemented with or without Arsenic. The arsenic tolerance profiles were compared with those of a German collection strain Exiguobacterium aurantiacum DSMZ 6208. Moreover, the presence of known resistance genes such as ACR3 or arsB as possible resistance mechanisms was assessed by degenerate oligonucleotides. In addition a proteomic approach using two-dimensional polyacrylamide gel electrophoresis (2D PAGE) was applied to investigate the tolerance to Arsenic of Exiguobacterium sp. S17.

All Exiguobacterium sp. tolerated As(V). Interestingly, the strain S17 was able to grow also in As(III). Accordingly the gene ACR3 was present only in this strain.Differences were also observed in the two-dimensional electrophoretic profiles of the S17 strain grown in As(V), As(III), and without arsenic.

This is the first report giving a highlight into the genetic mechanisms of Arsenic resistance by an Exiguobacterium sp. strain since is the only report to confirm the presence of the gene ACR3 in this species. The differences observed in the protein expression could provide further information about the bacterial mechanisms of Arsenic tolerance.

145A Prospection of novel proteases from a metagenomic library from Bertioga city mangrove sediments (São Paulo State - Brazil) using High Throughput Screening (HTS) Júlia Ottoni*1, Lilian Oliveira2, Débora Okamoto2, Luiz Neto2, Suzan Vasconcellos2, Anete Souza3, Valéria Oliveira1 1Campinas State University - UNICAMP / Division of Microbial Resources, Brazil, 2São Paulo Federal University - Unifesp, Brazil, 3Campinas State University - UNICAMP / Center of Molecular Biology and Genetic Engineering, Brazil

Mangrove is a peculiar ecosystem located on flood plains of the tides associated with estuaries, bays and lagoons and protected from wave action and ocean currents. Mangroves have an intense biological activity and plant and animal diversity and they also constitute, in general, an extreme

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habitat for microorganisms because of the high salinity, anoxic conditions, lack of nutrients, pH variations, among other adverse conditions. In this sense, microbial communities in mangroves are supposed to be adapted to such harsh conditions, and may constitute an interesting source of bioactive molecules. Researches involving microbial diversity in these environments are scarce, making the knowledge and exploitation of new microorganisms in mangroves an urgent issue. The aim of this work was to explore the potential of the microorganisms from mangrove sediments using metagenomic and high throughput screening tools to identify proteolytic activity, which are of great interest in industrial processes. A metagenomic library was constructed using Fosmids as vectors and the "Cloning-Ready Copy Control pCC2FOS" kit (Epicentre®), following the manufacturer's recommendations. A total of 672 clones were subjected to high throughput functional screening for proteases, based on the use of Fluorescence resonance energy transfer (FRET) peptide libraries probe. For this assay, clones were replicated to 96 well plates containing 160 mL of LB medium added of chloramphenicol (12.5 mg/mL) and the plates were incubated at 37ºC for 4 hours. After incubation, plates were centrifuged for 10 minutes at 4ºC 10.000 rpm and aliquots of 50 mL from the supernatant were added to 150 mL buffer solution (Tris 50 mM, pH 7.0) containing the library solutions (Abz substrate 5 mM). The hydrolysis of the FRET peptides was quantified using a Plate Spectrofluorimeter by measuring the fluorescence of Abz (ortho-aminobenzoic acid) as the fluorescence donor at lex 320 nm following emission at lem 420 nm and Q-EDDnp (glutamine-[N-(2,4-dinitrophenyl)-ethylenediamine]) as the fluorescence acceptor. The reactions were followed over 2 hours. Fifteen positive hits were obtained, which showed values above 0.009 of fluorescence intensity (cut off 0.008), demonstrating the proteolytic activity of the clones from mangrove sediments. The results obtained so far confirmed the potential of the microbiota from mangrove systems as a source for novel biocatalysts with proteolytic activity. Further steps of the work will comprise digestion of clones inserts and ligation into an expression vector for activity analysis and further characterization.

146A Bacterial abundances and phytopigments on sediments from Southwest Atlantic Rodolfo Paranhos*1, Anderson Cabral2, Fernando Pinto2 1Rio de Janeiro Federal University / Institute of Biology, Brazil, 2UFRJ / Institute of Biology, Brazil

Continental margins of the oceans play a key role in global biogeochemical cycles, and the microbiology of the sediments is poorly studied. In Bacterial abundance and phytopigments where investigate in sediments from the Southwest Atlantic Ocean, along the Campos Basin at Brazilian coast, and we study whether variables as water depth, sediment characteristics and productivity from upper layers could explain deep-sea sediment trophic conditions. The work was on the frame of Habitats Project - Campos Basin Environmental Heterogeneity by CENPES/PETROBRAS. Sediment samples were obtained in triplicates using a box-corer device, and sub-samples for bacterial and phytopigments were transferred to sterile tubes, and flash frozen in liquid nitrogen. At laboratory samples were thawed and (1) bacteria were extracted by pyrophosphate/sonication and then quantified by flow cytometry, while (2) pigments were extracted with acetone 100% and quantified by fluorimetry. Bacterial abundances decrease towards deep waters, ranging from 1,300x107 at 25 m to 3.6x107 cells.g-1 at 3,000 m, without differences between dry and rainy seasons. Chlorophyll a also was distributed with a clear vertical trend, with median values from 1.5 µg.g-1 at 25 m to <0.02 µg.g-1 deeper than 1,300 m. The values from rainy season on plataform (<150 m) were higher than those obtained during dry season, reflecting the continental contribution. At depths higher than 400 m very low chlorophyll values were observed, despite the season. Degradation products seems to have a different pattern, and higher values were obtained during dry season. Bacteria and phytopigments seem to be coupled the organic matter supply from surface waters and their productive cycles. The canyons were regions where high variability was observed for both bacteria and pigments, with higher values inside canyons than those obtained in its adjacent areas, indicating they are important for organic matter exportation towards deep provinces.

147A Experimental and single cell approaches to understanding microbial life in subsurface sediments underlying the extremely oligotrophic South Pacific Gyre Anand Patel*1, Tim G. Ferdelman2, Andreas Krupke2, Wiebke Ziebis1 1University of Southern California, United States, 2Max-Planck-Institute for Marine Microbiology, Germany

The South Pacific Gyre (SPG) is the largest oceanic province on our planet, yet very little is known about microbial life at and below the seafloor of this extremely oligotrophic region. In the better-studied

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continental margins, sediments have very different biological processes at play. Chlorophyll concentrations in the water column and carbon burial rates at the seafloor within the gyre are the lowest in the Ocean. Within the gyre, the seafloor is thinly draped with sediments comprised mostly of deep-sea red clay or nanofossil carbonate ooze (with water depths of 3500 to 5700 m and sediment thickness of ~70 m at the edges to < 2 m in the very center). During IODP Expedition 329 seafloor drilling, sediment cores were recovered along 2 transects at 6 sites spanning from the gyre edges to its ultra-oligotrophic center, plus a 7th site just to the south of the gyre. The main goal of the expedition was to explore the nature of the sub-seafloor habitats and the microbial communities of this vast ocean area. Onboard studies documented that oxygen and nitrate penetrated the entire sediment column within the gyre, whereas at the outside station oxygen was depleted within the top 1.5 m, exhibiting a mainly anoxic sediment column, typical of less oligotrophic ocean regions. Microbial cell abundances were very low within the gyre sediments, with values at the detection limit and became undetectable with greater depth. To explore and compare the metabolic activities of these entirely oxic sub-seafloor habitats, sediments samples were taken from drill cores at 3-4 different sediment depths over the entire thickness of the sediment cover at each site. We performed onboard incubation experiments, using stable and radio-isotopic labeled substrates, to test for potential autotrophic versus heterotrophic metabolisms (14C-carbon dioxide, 14C-acetate), as well as for nitrogen turnover and uptake (15N-dinitrogen, 15N-ammonium). A protocol was developed and tested to efficiently (80- 90%) extract and concentrate intact cells from these very low-abundance habitats for single cell analyses using nanoSIMS. Initial examination of our cell extracts, using FISH, indicated that bacterial cells dominate. NanoSIMS analyses showed strong incorporation of 15N-ammonium into bacterial cells. We are currently performing statistical comparisons to explore a possible trend of uptake rates across the gyre and with depth. The results of our investigations will provide a significant step towards understanding microbial life underneath oligotrophic open ocean gyres.

148A Nature of the microbial life in an Antarctic subglacial lake ecosystem David Pearce*1, Gavin Burns1, Dominic Hodgson1, Charles Cockell2 1British Antarctic Survey, United Kingdom, 2University of Edinburgh, United Kingdom

Antarctic subglacial lake ecosystems have the potential to be one of the most extreme and interesting environments on Earth, with combined stresses of high pressure, low temperature, permanent darkness, low-nutrient availability and variable oxygen concentrations, where the predominant mode of nutrition is most likely to be chemoautotrophic. Although direct exploration of subglacial lakes buried deep under the Antarctic Ice Sheet has yet to be achieved, at the retreating margins of the ice sheet, there are a number of locations where former subglacial lakes are emerging from under the ice that remain perennially ice covered. Here we present the results of a study of one of these lakes, Lake Hodgson (72° 00.549’S, 068° 27.708’ W) and describe the microbial life it contains. With three ambitious projects likely to access subglacial Antarctic lake ecosystems for the first time during the next two years, a unqiue opportunity now exists to address some fundamental scientific questions about the microbial ecology of these fascinating ecosystems.

149A Screening of biosurfactant producing bacteria from oil reservoir of Northern Petroleum Development Center, Chiang Mai Province Thanachai Phetcharat*1, Pinan Dawkrajai2, Sakunnee Bovonsombut1 1Chiang Mai University, Thailand, 2Northern Petroleum Development Center, Thailand

Microbial enhanced oil recovery (MEOR) is tertiary method purposed to increase oil recovery since the residue oil in the reservoir is two-thirds after primary and secondary production.. Biosurfactants are important substances in MEOR, which reduce surface tension in water solutions and hydrocarbon mixtures that properties made hydrocarbon or crude oil can mix in water. This study was conducted to find out the biosurfactants-producing bacteria from oil reservoir of Northern Petroleum Development Center for their In situ MEOR.

The 20 samples were collected from core and cutting of oil reservoir. Four media; nutrient broth, half nutrient broth, soil extract and tryptic soy broth were compared to isolate bacteria at 50°C. The only facultative anaerobic bacteria were selected. The isolated were purified by using the conventional method. The Drop-collapse method was used to detect biosurfactants produce by bacterial isolated from soil in oil well. The biosurfactant producing bacteria were compared with their community in the

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original soil using PCR-DGGE method. The isolates were identified by using API and 16S rRNA analysis.

Sixty pure cultures were isolated from both core and cutting of the reservoir. Most of them are gram positive, rod shape and spore-forming. The result showed that 3 isolated bacteria have biosurfactants. The PCR-DGGE method indicated that they are among microbial community in oil sandstone. The 16S rRNA analysis showed all 3 isolated were Bacillus licheniformis.

The results showed that application of biosurfactant producing bacteria in MEOR to increase crude oil by indigenous microorganisms should be possible. However, the condition suitable for their growth and biosurfactants production in the borehole should be further studied.

150A Molecular and microbial analysis of diversity present in unpreserved wood from King Henry VIII's Tudor warship the Mary Rose Joanne Preston*, Joy Watts University of Portsmouth, United Kingdom

The Mary Rose Tudor warship provides a unique environment for research. Submersion and burial in anoxic marine sediment for almost 500yrs has favoured reductive cycling of iron and sulfur compounds in the Mary Rose hull and her associated artefacts. Consequently, timbers of the Mary Rose hull contain approximately 2 mass % reduced sulfur and iron compounds. The microbial community associated with archaeological timbers under different states of preservation have been characterized using both molecular and culture-based techniques. We present the microbial diversity of an unpreserved section of Mary Rose wooden stem post, raised in 2004. This stem post section historically contained an iron bolt, and the wood surrounding the bolt hole was degraded and covered in yellow deposits. The 16S rRNA sequence based clone library analysis indicates the presence of iron cycling bacteria. Sequences of 97% similarity to the acidophile iron and sulphur oxidising Gammaproteobacteria Thiobacillus prosperus and iron reducing Alphaproteobacteria Acidiphilium species were present in the clone library. The Thiobacillus prosperus-like strains identified in the archaeological wood have proven as yet, unculturable. Acidophilic, heterotrophic iron and sulfur oxidizing Alicyclobacillus strains have been enriched from the Mary Rose stem post in co-culture with Mary Rose Acidiphilium strains, and subsequently isolated in pure culture. The ongoing characterisation of a halotolerant, spore forming, Alicyclobacillus iron and sulfur oxidising strain, is presented here. The clone library of the unpreserved stem post was, however, dominated by sequences of 95.6-93.5% similarity to the gas vacuolated gliding bacterioidetes Meniscus glaucopis and other related species of bacteria from the Sphingobacteria and Cytophaga. This research offers insight into the bacterial population resident in waterlogged wood buried in marine anoxic sediments.

151A Study of interactions copper-microorganisms in impacted marine Chilean sediments Laurent Quillet1, Ludovic Besaury*1, Gerard Muyzer2 1UMR CNRS 6143, France, 2Laboratory of Microbiology, University of Delft, Netherlands

Copper is an element essential for numerous microorganisms but can be considered as a toxic element at very high concentrations. Sediment samples were taken from the outlet of Canal Palito (Northern Chile), a copper mine wastewater discharge channel that flows into the Pacific Ocean at the beach of Caleta Palito, Chile. Caleta Palito was a tailing deposit from 1975 to 1989 receiving more than 130,000 million metric tons of untreated copper mining wastes, which were never removed and present an average copper concentration of 800 ppm. A site closed to Caleta Palito named Flamenco presenting no copper-contamination was used as reference site.

Molecular tools were developed to study copper-resistance genes copA and cusA encoding respectively for an ATP-ase type IB pump oxidase proteins and a Heavy-MEtal Resistance NoDulation cell protein (HME-RND). Abundance of these two genes was quantified and reveals an important augmentation of this copper-resistance gene regarding the augmentation of copper concentration. Results of quantification of those two genes showed that gene copA was always more abundant than gene cusA, whatever copper concentration or oxic and anoxic conditions. Cloning of two genes was realized and suggested that copper leads to an strong shift of diversity of those two genes regarding to copper concentration.

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More attention was focused on the microbial diversity present in both sites. Abundance and activity of bacterial and archaeal communities were studied in the non-impacted and impacted sediments. Results of 16SrDNA and 16SrRNA quantification showed that Bacteria were more abundant than Archaea in the contaminated site whereas surprisingly Archaea were more active suggesting a better adaptation to metal toxicity. Generally activities of both communities are always higher in the contaminated site than in the uncontaminated one.

Fingerprinting techniques of 16SrDNA and 16SrRNA demonstrated a modification of structure of bacterial communities in those two environments, and such results were confirmed by clone libraries. Bacterial active communities in the contaminated site were highly dominated by members from Alphaproteobacteria and Gammaproteobacteria. Clones libraries of 16SrRNA of Archaea revealed that members of Euryarchaeota are associated with high levels of contamination.

A culturable study was also realized to study microorganisms able to grow in such impacted environments both in oxic and anoxic conditions. Results showed that bacteria belonging to Bacillus sp. and to sulfate-reducing bacteria as Desulfovibrio sp. were both dominant respectively in oxic and in anoxic conditions and presented high levels of resistance to copper until 400ppm. Abundance and diversity of sulfate-reducing bacteria (SRB) determined by molecular tools showed a decrease of the abundance of SRBs and a modification of diversity according to the copper concentration, suggesting an adaptation process of a specific SRB community. No sulfate reducing activity was observed in highly impacted sediments suggesting that SRBs are not involved into copper cycle by sulphide-copper ions precipitation at high concentration of copper.

151B Abundance, activity and diversity of bacterial and archaeal communities in highly copper-contaminated chilean marine sediment Laurent Quillet*1, Besaury Ludovic2, Ghiglionne Jean-Francois3 1UMR CNRS 6143, France, 2UMR CNRS 6143 Université de Rouen, France, 3UMR CNRS 7621, Laboratoire d'Océanographie Microbienne LOMIC, France

Marine sediments are considered as sink for heavy metals and in those ecosystems, copper is described as an important pollutant due to its toxicity to the three domains of life: Bacteria, Archaea and Eukarya. Many publications were interested in the impact of copper to bacterial communities but only few dedicated to study the abundance and activity of bacterial and archaeal communities in contaminated sediments.

We have studied by molecular tools the impact of copper on active bacterial and archaeal communities in a copper mine wastewater channel that discharged from 1975 to 1989 130,000 million metric tons of untreated copper mining wastes. 2 cores of approximately 25 cm were collected in october 2009 from 2 sites presenting different copper concentrations. The first site called Flamenco is uncontaminated (average of 3 ppm) and the second one, Palito, is highly contaminated (average of 850 ppm).

Total nucleic acids were extracted and abundance and activity of bacterial and archaeal communities were determined by real time PCR. Results showed that abundance of bacterial and archaeal communities are 2 to ten times more abundant in the uncontaminated sediment than in the contaminated one. Abundance of Bacteria is always higher than abundance of Archaea whatever the studied site; nevertheless, compared to Bacteria, a superior proportion of Archaea was found in Palito’s core, suggesting a possible better adaptation of this domain. Such possible adaptation was verified by quantifying activities of those two communities and confirmed that archaeal communities are more active in the highly contaminated sediment than in the uncontaminated one, and also an activity superior to that observed for the bacterial community. 16SrDNA and 16SrRNA CE-SSCP were performed to study the structure of bacterial communities in the two cores and revealed an important shift, certainly caused by the augmentation of copper and confirmed an adaptation of those communities in Palito’s core. Clone libraries of 16S rRNA transcripts were realised to study the diversity of the two microbial active communities and revealed that microorganisms from Euryarchaeota are predominant in high levels of contamination for archaeal communities and from Alphaproteobacteria and Gammaproteobacteria.

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152A Resistance of biological sand crust organisms to excess light during desiccation Hagai Raanan*, Yitzhak Ohad, Nir Keren, Aaron Kaplan The Alexander Silberman Institute of Life Sciences The Hebrew University, Israel

Biological soil crusts play an important role in stabilizing sandy areas and can influence the biotic composition of deserts. Destruction of these crusts is considered an important promoter of desertification in arid and semi-arid regions. The crusts are formed by the adhesion of the sand to extracellular polysaccharides (EPS) secreted mostly by filamentous cyanobacteria (includingNostoc,Microcoleus, ChroococcidiopsisandGloeocapsa). These organisms, the main primary producers in biological desert crusts, are able to acclimate to extreme temperatures, excess light and frequent hydration/dehydration cycles. The mechanisms involved, however, are largely unknown. It is likely that the ability to activate metabolism and grow when water is available and to shutdown metabolic activity during dehydration plays an important role in this acclimation. Our recent study showed that activation of photosynthesis during hydration of a biological crust, consisting mostly of Microcoleussp., did not requirede novoprotein synthesis. Efficient energy transfer to the photosynthetic reaction centers and electron transfer activities were observed within a few minutes following hydration.

Of particular importance are the mechanisms whereby these desiccation-tolerant cyanobacteria activate the photosynthetic system upon hydration and protect themselves against photoinhibition under excess light, particularly during dehydration. It is well-established that photosystem II is highly susceptible to photoinhibition and is accompanied by rapid degradation of its core proteins (including D1 and D2). Coordination of light energy flux to the reaction centers with the rate of electron transport and CO2 fixation is extremely important, particularly during dehydration; otherwise, photodynamic damage of the photosynthetic machinery may occur.

In order to uncover protective mechanisms from excess light during desiccation, we compared the response to excess light of several desiccation-tolerant cyanobacteria with well-studied freshwater cyanobacteria. Measurements were taken of oxygen evolution and fluorescence parameters on crusts and isolated strains from the sand dunes of Nizzana, NW Negev, grown on sand or liquid media.

Surprisingly our results showed much less resistance of the desiccation-tolerant cyanobacteria to excess light as compared to freshwater cyanobacteria. The desiccation-tolerant cyanobacteria also showed a rapid decrease in Fv/Fm and faster Qa reoxidation after high light treatment. These results and others suggest that photosynthetic activity of the cyanobacteria in the crust occurs at low light levels and that protection mechanisma are activated as soon as direct sunlight hits the crust. This protection mechanism may involve a cyclic electron flow within PSII.

153A Carbon dioxide affects growth and thiosulphate reduction of thermophilic Petrotoga sp. Jana Rakoczy*1, Claudia Gniese2, Hans-Hermann Richnow3, Axel Schippers1, Martin Krüger1 1Federal Institute for Geosciences and Natural Resources, Germany, 2TU Bergakademie Freiberg, Germany, 3Helmholtz Centre for Environmental Research - UFZ, Germany

Carbon capture and storage (CCS) is considered a promising new technology which is able to reduce carbon dioxide emissions into the atmosphere and thereby decelerate global warming. During CCS, carbon dioxide is captured from emission sources (for example fossil fuel power plants or other industries), pressurised, and finally stored in deep geological formations, such as former gas or oil reservoirs as well as saline aquifers. However, with CCS being a very young technology, there are yet a number of unknown factors that need to be investigated before declaring CCS as being safe and risk-free. Our research investigates the effect of high carbon dioxide concentrations and pressures on indigenous microorganisms that colonize potential storage sites.

Growth experiments were conducted using the thermophilic thiosulphate-reducing bacterium Petrotoga sp., isolated from formation water of the gas reservoir Schneeren (Lower Saxony, Germany), situated in the Northern German Plain. Growth (OD600) and activity (sulphide formation) were monitored at different carbon dioxide concentrations (50% and 100% in the gas phase) at atmospheric pressure, and at 1.5 bar overpressure, and were compared to control cultures grown with 20% carbon dioxide. Cultivation with high carbon dioxide concentrations at atmospheric pressure only slightly affected cell growth or thiosulphate-reducing activity during the first cultivation period (10

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days), whereas long-term cultivation at 1.5 bar reduced both maximum OD600 and sulphide formation suggesting a more pronounced effect with prolonged cultivation already at slight overpressure.

In current experiments, cultivation with supercritical carbon dioxide at 70 bar will further examine physiological and molecular properties of the model organism allowing for prediction of sensitivity and/or adaptability of reservoir microbial communities in potential future storage sites.

481B The effect of hydrostatic pressure on biodegradation of a mixture of benzene, toluene, ethylbenzene and o-xylene Kristin T. Ravndal*, Andrea Bagi, Roald Kommedal University of Stavanger, Norway

The oil and gas industry continue to expand, and exploitation of hydrocarbon resources is moving into the deep-sea. The Deepwater Horizon oil spill and the possibility of future oil spills in this part of the ocean enhance the importance of studying the fate of oil released there. One of the processes important in removal of organic pollutants from aquatic environments is biodegradation.

In this work two sets of experiments were performed at 1, 80, 170 and 340 bar to assess the effect of pressure on biodegradation of a mixture of benzene, toluene, ethylbenzene and o-xylene (BTEX) in seawater. Substrate removal was analysed using gas chromatography, and in the first set of experiments prokaryotic community composition was analysed with denaturing gradient gel electrophoresis at the start and endpoint of the experiment.

Analysis of substrate removal showed a similar trend in both sets of experiments. Degradation of all four substrates was slower under a higher hydrostatic pressure. In the first experiment substrate removal was measureable at 1 and 80 bars, while no degradation was observed at 170 bar and 340 bar. The lag phase was shorter at 1 bar compared to at 80 bar. In the second experiment degradation occurred at 1, 80 and 170 bar, but no degradation was detected at 340 bar even after 221 days. There was no significant difference between the lagphase at atmospheric pressure and at 80 bar, while the lagphase at 170 bar was notably longer than at the two lower pressures.

Genetic fingerprints of 16S rRNA gene amplicons analysed with denaturing gradient gel electrophoresis indicated that prokaryotic diversity decreased as a result of BTEX exposure. Moreover the band patterns observed at 340 bar appeared to be different from patterns found in samples that were incubated at lower pressures. This indicates that hydrostatic pressure indeed affects which microorganisms will be able to grow and degrade the most soluble hydrocarbons abundant in crude oil.

Measurements of substrate removal in this research indicate that high hydrostatic pressure slows down bacterial degradation of BTEX. This effect is evident from experiments performed at 170 bar and 340 bar, but not from the tests at 80 bar. As these pressures (80 and 170 bar) correspond to depths of 800 and 1700 meters, our results indicate that pressure effect will become significant between these depths.

154A Fungal diversity from deep marine subsurface sediments (IODP 317, Canterbury Basin, New Zealand) Vanessa Redou*1, Danielle Arzur2, Gaëtan Burgaud2, Georges Barbier2 1Laboratoire Universitaire de Biodiversité et Ecologie Microbienne (EA3882), France, 2Laboratoire Universitaire de Biodiversité et Ecologie Microbienne (EA3882), IFR148 ScInBioS, Université Européenne de Bretagne, Université de Brest, ESMISAB, Technopôle de Brest Iroise, 29280 Plouzané, France

Recent years have seen a growing interest regarding micro-eukaryotic communities in extreme environments as a third microbial domain after Bacteria and Archaea. However, knowledge is still scarce and the diversity of micro-eukaryotes in such environments remains hidden and their ecological role unknown. Our research program is based on the deep sedimentary layers of the Canterbury Basin in New Zealand (IODP 317) from the subsurface to the record depth of 1884 meters below seafloor. The objectives of our study are (i) to assess the genetic diversity of fungi in deep-sea sediments and

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(ii) identify the functional part in order to better understand the origin and the ecological role of fungal communities in this extreme ecosystem.

Fingerprinting-based methods using capillary electrophoresis single-strand conformation polymorphism and denaturing high-performance liquid chromatography were used as a first step to raise our objectives. Molecular fungal diversity was assessed using amplification of ITS1 (Internal Transcribed Spacer 1) as a biomarker on 11 samples sediments from 3.76 to 1884 meters below seafloor.

Fungal molecular signatures were detected throughout the sediment core. The phyla Ascomycota and Basidiomycota were revealed with DNA as well as cDNA. Most of the phylotypes are affiliated to environmental sequences and some to common fungal cultured species.

The discovery of a present and metabolically active fungal component in this unique ecosystem allows some interesting first hypotheses that will be further combined to culture-based methods and deeper molecular methods (454 pyrosequencing) to highlight essential informations regarding physiology and ecological role of fungal communities in deep marine sediments.

155A The microbial ecology of cementitious radioactive waste Athanasios Rizoulis*1, Adam Williamson1, Christopher Boothman1, Antoni Milodowski2, Katherine Morris1, Jonathan Lloyd1 1University of Manchester, United Kingdom, 2British Geological Survey, United Kingdom

The proposed concept for intermediate level radioactive waste disposal in the UK is based on a multi barrier system containing cementitious wasteforms that will be placed in a deep geological repository and potentially backfilled with a cementitious backfill. The highly alkaline conditions that will develop when the host environment becomes saturated are intended to minimise radionuclide solubility and thus the risk of radionuclide transport to the biosphere. However, the biogeochemical gradients that will develop across the interface between the alkaline deep cementitious geological disposal facility and the geosphere are poorly understood in terms of their impact on radionuclide solubility. In addition, the excess of iron, metal oxides, nitrate and radionuclides and the availability of organic carbon in intermediate level waste is likely to create conditions favourable for microbial growth. Microbial transformations of radionuclides under reducing conditions are well studied at circumneutral pH, but very little is known about microorganisms that may interact with metals and radionuclides under alkaline conditions, although they may potentially control the speciation and solubility of several key radionuclides and therefore will be critical in underpinning any safety case.

In this study, samples from two highly alkaline environments, that are analogous to concrete-based intermediate level waste, were used to understand the microbial transformations potential of these systems to affect metals and radionuclides at high pH. In samples taken from the hyperalkaline (pH up to 11.7) Allas Springs in Cyprus, diverse bacterial communities were detected by 16S rRNA gene cloning and sequencing. Anoxic laboratory microcosms amended with organic carbon and Fe(III)-citrate showed the potential of some of these samples to reduce soluble Fe(III) at pH 10, while pure cultures of Fe(III)-reducing bacteria were also isolated on solidified media at pH 10. Further microcosms were set up using sediment samples from another highly alkaline environment, the tailings of a legacy lime works site at Harpur Hill, Buxton, UK. These microcosm experiments have shown the capacity of indigenous microbial populations to reduce the amorphous Fe(III) mineral ferrihydrite to magnetite, as well as U(VI) to uraninite, at pH 10. Ongoing work with selected isolates from both experimental sites will investigate the impact of microbial metabolism on radionuclide behaviour at high pH. In a separate experiment, the pH limit for microbial growth in sediment microcosms amended with different electron acceptors was also investigated. These results indicate a clear cascade of electron acceptor utilisation in the order of nitrate > soluble Fe(III) > insoluble amorphous Fe(III) > sulfate, and that microbial activity is observed at up to pH 11 but not at pH 12 or above, in agreement with thermodynamic calculations. These results will be discussed in the context of the potential of alkaliphilic microorganisms to colonise and influence the evolution of cementitious geological repositories containing radioactive waste.

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156A Microbial diversity of lithifying communities in the Great Salt Lake, Utah Giovanni Rompato*1, Stephen Callister2, Jacob Parnell3 1Utah State University, United States, 2Pacific Northwest National Laboratory, United States, 3National Ecological Observatory Network, United States

The earliest evidence of life on Earth comes from fossils of lithifying microbial communities (stromatolites) dated to approximately 3.5-4 billion years ago. From that time until now, stromatolites have played an important role throughout Earth's history: they have driven carbon sequestration, and nutrient cycling during the evolution of life, and are responsible for the dealcalinization of the oceans and the formation of the current atmosphere. The study of modern analogs of ancient communities can provide insight to the development of microbial metabolic pathways and community interaction that can help shed light on the evolution of biogeochemical cycles. The GSL has one of the largest and least studied assemblages of living stromatolites in the world and is an invaluable resource for studying lithifying microbial communities. In this study we used 16S pyrosequencing to study the microbial community composition of a stromatolite collected from the hypersaline South Arm (15% salt) and North Arm (30% salt) of the Great Salt Lake (GSL), Utah, USA. Stromatolites were collected from the South and North Arms of the GSL during the summer of 2011, DNA was extracted using a MoBio Soil DNA extraction Kit. The V4 of the bacterial 16S rDNA gene and archeal equivalent was amplified and sequenced on a Roche 454 using the standard amplicon sequencing protocol. In the South Arm stromatolites we found representatives of the same functional groups that have been described in most modern lithifying microbial communities (cyanobacteria, anoxygenic phototrophs, aerobic heterotrophic, fermenters, anaerobic heterotrophs and sulfide oxidizing bacteria) in Shark Bay, Australia and Exuma Sound in the Bahamas. We also found several Archea species present in the microbial community. In contrast, the North Arm stromatolite community is dominated by Archea with few bacteria species (Salicola Sp. and Salinibacter ruber) present. Futhermore, a comparison of initial peptide features measured by LC-MS also suggests differences in the stromatolites proteomes from the two different locations. These results indicate that the general community structure of GSL South and North Arm stromatolite communities is quite different, with regard to functional taxonomic groups, to other stromatolites communities described before. However, we found differences in community members at the family level that suggest that functional roles are maintained by interchangeable family members depending on prevailing environmental conditions. For example, primary production in the GSL South Arm stromatolites is performed by cyanobacteria (Oscillatoria sp. and Phormidiaceae sp.) that have been shown to tolerate high salt concentration, while in the North Arm stromatolites the same functional role seems be performed by Archea.

157A Liberation of microbial substrates from macromolecular organic matter by non-supercritical CO2 Patrick Sauer*, Clemens Glombitza, Jens Kallmeyer University of Potsdam, Institute of Earth and Environmental Science, Germany

The worldwide search for suitable underground storage formations for CO2 also considers coal-bearing strata. CO2 is already injected into coal seams for enhanced recovery of coal bed methane. However, the geochemical and microbiological effects of increased CO2 concentrations on organic matter rich formations are rarely investigated.

The injected CO2 will dissolve in the pore water, causing a decrease in pH and resulting in acidic formation waters. Low molecular weight organic acids (LMWOAs) are chemically bound to the macromolecular matrix of sedimentary organic matter and may be liberated by hydrolysis, which is enhanced under acidic conditions. Recent investigations outlined the importance of LMWOAs as a feedstock for subsurface microbial life [Glombitza et al. 2009]. Therefore, injection of CO2 into coal formations may result in enhanced nutrient supply for subsurface microbes.

To investigate the effects of highly CO2-saturated waters on the release of LMWOAs from coal, we developed an inexpensive high-pressure high temperature system that allows manipulating the concentration of dissolved gases up to 60 MPa and 120°C, respectively. The sample is placed in a flexible, gastight and inert PVDF sleeve, separating it from the pressure fluid and allowing for subsampling without loss of pressure.

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Lignite samples from the DEBITS-1 well, Waikato Basin, NZ and the Welzow-Süd open-cast mine, Niederlausitz, Germany, were extracted at 90° C and 5 MPa, with either pure water, CO2-saturated water, CO2/NO2 or CO2/SO2-saturated water. Subsamples were taken at different time points during the 72 hrs. long extraction.

Extraction of LMWOAs from coal samples with our pressurised system resulted in yields that were up to four times higher than those reported for Soxhlet extraction [Vieth et al. 2008]. These higher yields may be explained by the fact that during Soxhlet extraction the sample only gets into contact with freshly distilled water, whereas in our system the extraction fluid is circulated, resulting in more acidic extraction conditions.

In comparison to pure water extractions, CO2-saturated water affected the extraction yield in both directions by up to 40 percent. For the lignite from DEBITS-1 well, CO2-saturated water resulted in a permanently lower yield, whereas the lignites from the Lausitz showed an increase in formate and a decrease in oxalate.

LMWOAs found in the extraction fluid may not just result from hydrolysis but also from different secondary reactions. It was suggested that oxalate in aqueous extracts of coals is a result of the decomposition of 1,2-dihydroxy-carboxylic acids [Bou Radd et al. 2000]. We assume that for oxalate (and maybe for other LMWOAs as well) the extraction yield is not only affected by hydrolysis but also by secondary reactions, which may be suppressed in the presence of CO2 and other gasses dissolved in the extraction medium.

These results show the importance of performing laboratory simulations of subsurface processes under conditions that resemble the true in-situ conditions as closely as possible.

158A Bacterial communities established in bauxite residues with different restoration histories Achim Schmalenberger*1, Orla O'Sullivan2, Jacinta Gahan1, Paul D. Cotter2, Ronan Courtney1 1University of Limerick, Life Sciences, Ireland, 2Teagasc, Moorepark Food Research Centre, Ireland

Bauxite residue is the alkaline by-product generated when alumina is extracted from bauxite ores. Its production is estimated at about 120 Mt/a and these residues are commonly deposited in nearby impoundments. These sites represent hostile environments with increased salinity and alkalinity and little prospect of re-vegetation when left untreated. While re-vegetation efforts on bauxite residue are now better studied, virtually nothing is known about concurrent establishment of a microbiota, possibly similar to soil communities. Here we report the establishment of below ground bacterial communities in two types of bauxite residue amendments (compost and gypsum addition, re-vegetation) in comparison to a non-amended bauxite residue.

Bauxite residues were deposited in 1993 in County Limerick, Ireland. Re-vegetation took place in 1997 with 120kg/ha compost and 45kg/ha gypsum amendment (J site), in 1999 with 120kg/ha compost and 90kg/ha gypsum amendment (R site) or residues were not treated (M site). Samples were taken in 2009 and 2011 from all three sites in mixed samples taken from 0-10 cm depth in triplicates. DNA extraction was carried out and 16S rRNA genes were amplified to conduct DNA fingerprint analysis and to generate 16S amplicons in a 454 FLX pyrosequencer. Operational taxonomical units were clustered with a similarity cut off at 97% and diversity analysis was calculated using Qiime. Taxonomic analysis of sequences was implemented with a combination of BLAST and MEGAN.

Fingerprint analysis from samples taken in 2009 and 2011 identified clear separations of the microbial communities in the non-amended and the amended sites and identified differences between the two amended types. The determined alpha diversity of the non-amended site was estimated (chao1) to be in the region of 332 to 608 while the alpha diversity of the R and J series varied between 900 and 19000. Hierarchical clustering separated the M site from the amended treatments (J, R). However, the separation between the 2 amendment types was less pronounced than the separation by sampling year (2009 and 2011). The taxonomic analysis revealed that the remediation resulted in the accumulation of bacterial populations typical for soils that include high numbers of Verrucomicrobia (e.g. Spartobacteria), Acidobacteria (e.g. Acidobacteriaceae) and Proteobacteria (e.g.

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Nitrosomonadaceae). While higher numbers of Proteobacteria (e.g. Beijerinckiaceae) and Verrucomicrobia (Opitutaceae) were also present in the non-amended site several other taxonomic groups dominated this site such as the Rickettsiales, Propionibacteriaceae and Chitinophagaceae.

While the compositional analysis shows that a substantial bacterial diversity exists in the initially sterile bauxite residue, the beta diversity and the taxonomic analysis confirm that the lack of treatment prevented a development of the site towards a semi-natural soil like environment. In contrast, both amendment treatments created diverse soil like bacterial communities alongside diverse vegetation on the surface. These findings suggest that bauxite residues can be transformed into sustainable semi-natural like environments in temperate climates while non-amended bare sites may not develop towards a soil like state over twice the time.

159A Environmental controls on microbial diversity in geothermal springs Christine Sharp*1, Allyson Brady1, Joongjae Kim1, Stephen Grasby2, Matthew Stott3, Peter Dunfield1 1University of Calgary, Canada, 2Geological Survey of Canada, Canada, 3GNS Science, New Zealand

Extreme environments (e.g. high temperature, salinity or pH extremes) are a large reservoir of uncultivated microbial biodiversity. It is commonly understood that pH and temperature are important controls on microbial diversity. However, despite extensive study of the microbial communities in extreme pH and temperature environments, the controls of overall diversity have not been explicitly tested. The objective of this study was to use 16S rRNA gene pyrotag sequencing to determine the diversity of bacterial communities in a large sample set of geothermal springs spanning a wide range of pH and temperature. Soil and sediment samples were collected at various times of the year between fall 2009 and spring 2012 from geothermal springs in western Canada (British Columbia and Alberta) and New Zealand, representing a temperature range of ~4 to 86°C and a pH range of ~1.8 to 9.0. 16S rRNA pyrotag sequence analysis was performed on over 100 sediments, soils and mats from 26 distinct geothermal springs and results were analyzed using the QIIME pipeline. The filtered data set included over 700,000 sequences with an average of ~7000 reads per sample. To our knowledge this is the largest pyrosequencing data set that examines both pH and temperature gradients in a single study. Analysis of the Shannon index, Faith's index of phylogenetic diversity (Faith's PD) and species richness estimators (OTUs detected and Chao1) suggested that diversity decreases as pH and temperature become more extreme. Peak diversity was observed in the pH range of 6.5 to 8.5 and temperature from 20 to 30°C. Hot, acidic sites showed the lowest diversity. The archaeal phyla Euryarchaeota and Crenarchaeota dominated spring samples below pH5, while the Crenarchaeota dominated at pH9. Proteobacteria were found in high abundance at all pH's below pH9. Regression analysis showed that pH and temperature alone accounted for 59% of the total variance in microbial diversity (50% of species richness) across geothermal springs. Temperature was the strongest predictive variable alone, explaining 32% of the variability in diversity. pH alone explains 27%. Principle Coordinate Analysis (PCoA) using the Binary Jaccard and Bray-Curtis distances as well as the phylogenetic-based matrix UniFrac (weighted and unweighted) showed that the first dimension separates all acidic springs from neutral springs (both hot and cold) in the first dimension; and the second dimension separates cold from hot (>30°C) neutral springs. The data indicate that both pH and temperature play an important role in determining microbial diversity in geothermal springs on a global scale.

160A Microbial synthesis and transformation of dissolved organic matter in glacial environments Heidi Smith*1, Christine Foreman1, Rachel Foster3, Diane McKnight4 1Montana State Universtiy, United States, 2Max-Planck Institute for Marine Microbiology, Germany, 3INSTAAR, University of Colorado, United States

The Cotton Glacier stream, Antarctica, is a supraglacial stream chosen specifically for an extensive investigation of contemporary formation and storage of carbon in glacial ice. Dissolved organic matter (DOM) in this system increases seasonally from the growth of newly established microbial communities. DOM from glaciated systems is of importance because it contributes to the growing carbon pool in marine and freshwater aquatic environments, thereby also playing a role in the global carbon cycle. The Cotton Glacier fluvial water DOM is characterized by unique signatures unlike any

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DOM derived from freshwater or marine systems globally, including the previously well-studied lakes and streams of the McMurdo Dry Valleys, Antarctica. Fluorescence spectroscopy and XAD purification confirmed that the DOM from the Cotton Glacier is of microbial origin and lacks humic substances. The Cotton Glacier stream environment has an active microbial assemblage. Bulk uptake rates for primary production range from 1.079-4.750 µgC l-1 d-1 using 14C-carbonate/bicarbonate incorporation. Bacterial production ranges from 303-675 ng C l-1 d-1 measured using tritiated leucine incorporation. Chlorophyll a concentrations range from 0.3 to 0.53 mg l-1, and bacterial abundances from 2.94x10-4 – 4.5 x 10-5 cells ml-1. The dominant microbial assemblages in the Cotton Glacier stream are from Cytophagales and β-Proteobacteria lineages based on 16S rRNA sequence homology.

Understanding the transfer of carbon and nutrients between autotrophs and heterotrophs is essential to further our understanding of biogeochemical cycling throughout an ecosystem. In marine and freshwater aquatic environments the production of DOM by extracellular release of photosynthetic exudates by phytoplankton has the potential to contribute up to half of the carbon required to support bacterial growth. Using Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-nanoSIMS) we identified which populations took up specific tracers was well as the heterotrophic uptake of released exudates. Using this method we will directly measure uptake rates and estimate doubling time among different microbial members. Quantification of carbon transfer rates from phytoplankton to heterotrophic bacteria provides information linking the exudates and associated microbial processing to the global carbon pool.

161A Carnobacterium iners sp. nov., a psychrophilic, lactic acid-producing bacterium from the littoral zone of the Forlidas pond (Pensacola mountains), Antarctica Isabel Snauwaert*1, Bart Hoste1, Katrien De Bruyne2, Karolien Peeters1, Luc De Vuyst3, Anne Willems1, Peter Vandamme1 1Ghent University, Belgium, 2Applied Maths, Belgium, 3Vrije Universiteit Brussel, Belgium

Recently, the heterotrophic bacterial diversity in a microbial mat sample originating from the littoral zone of a continental Antarctic lake (Forlidas Pond) was examined. A large number of bacteria were isolated and characterized through repetitive element palindromic PCR and phylogenetic analysis of partial 16S rRNA gene sequences. Thirty lactic acid bacteria belonging to two main rep-clusters remained unidentified. Therefore, these isolates were included in a poly phasic taxonomic study involving phylogenetic analysis of the full 16S rRNA genes and multi-locus sequence analysis of the pheS, rpoA, and atpA genes. The obtained results proved that one strain, designated LMG 26641, belongs to Carnobacterium alterfunditum and that the other strain, designated LMG 26642T, can be assigned to a novel species with Carnobacterium funditum as its closest phylogenetic neighbor (99.2% 16S rRNA gene sequence similarity towards the type strain). DNA-DNA hybridization experiments confirmed that the latter strain represents a novel species, for which we propose the name Carnobacterium iners sp. nov., with strain LMG 26642T (= CCUG 62000T) as the type strain. Carnobacterium iners sp. nov. can be distinguished biochemically from other Carnobacterium species by the lack of acid production from carbohydrates.

162A Microbial degradation of the pesticide 2,4-D on the Greenland Ice Sheet Marek Stibal*1, Jacob Bælum1, William E Holben2, Sebastian R Sørensen1, Carsten S. Jacobsen1 1Geological Survey of Denmark and Greenland, Denmark, 2University of Montana, United States

The Greenland ice sheet (GrIS) receives organic carbon (OC) of anthropogenic origin from the atmosphere and/or local sources, including pesticides, and the fate of these compounds in the ice is currently unknown. The ability of heterotrophic microbes living on the ice surface to mineralise different types of OC is likely to be a significant factor determining the fate of anthropogenic OC on the GrIS. In this study we determine the potential of the microbial community from the surface of the GrIS to mineralise the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). It is one of the most easily degraded compounds among the phenoxyacetic acid herbicides, and the ability to mineralise 2,4-D has been found to be widespread in microbial communities around the globe. Functional genes involved in the degradation pathway have also been characterised. Thus, 2,4-D represents a very suitable model compound to use in order to gain an insight into pollutant degradation dynamics in natural environments, including those in the rapidly changing Arctic region.

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Surface ice cores were collected on the GrIS and incubated for up to 529 days in microcosms simulating in situ conditions. Mineralisation of side-chain- and ring-labelled 14C-2,4-D was measured in the samples and quantitative PCR targeting the tfdA gene, encoding an enzyme catalysing the first step in the degradation pathway of 2,4-D, in the DNA extracted from the ice after the experiment was performed.

We show that the supraglacial microbial community on the GrIS is of low diversity, but contains microbes capable of degrading 2,4-D. The low diversity of the community and the similarity of the detected clones to those from other icy environment clones suggest that the bacterial community on the GrIS is selected from a pool of propagules deposited on the surface of the ice sheet, based on the level of adaptation to the conditions in the surface ice. The 2,4-D degraders are likely present in very low numbers, and they can mineralise 2,4-D at a rate of up to 1 nmol per m2 per day, equivalent to ~26 ng C m-2 d-1.

Thus, the GrIS should not be considered to be a mere reservoir of all atmospheric contaminants, as it is likely that some deposited compounds will be removed from the system via biodegradation processes before their potential release due to the accelerated melting of the ice sheet

163A Physiological and genomic characterization of microorganisms in actively serpentinizing The Cedars springs Shino Suzuki-Ishii1, J. Gijs Kuenen*2, Kira Schipper2, Suzanne van der Velde2, Shunichi Ishii1, Greg Wanger1, Kenneth H. Nealson1 1J. Craig Venter Institute, USA, 2Delft University of Technology, Netherlands\

The ultra-basic, highly reducing springs and aquifers of the actively serpentinizing Cedars peridotite in coastal Northern California are so extreme as to challenge current dogma about the requirements for sustaining microbial life. Geochemical modeling of the springs has yet to reveal niches where described strategies for life can exist; however here life has found a way. Thus, understanding the strategies employed by these microorganisms may provide new insights into unknown mechanisms of microbial physiology and biochemistry and may shed light on life on early Earth ultramafic rocks and serpentinization were far more common than they are today. We report a culture independent survey of the multi-domain microbial diversity in the Cedars springs and seasonal community transition. The Cedars springs are characterized by some of the most extreme environmental and nutritional conditions on the planet; very high pH (11.50 - 11.85), extremely low Eh (< –600 mV), low ionic concentrations (≤ 2 mM Na+ , K+, Cl-), no detectable phosphate, nitrate, nitrite, or ammonium, low amounts of trace metals, and relatively high concentrations of Ca2+ (1-5 mM). In addition, the water contains H2 and CH4, representing a very significant energy source, however these waters contain neither oxygen, only very low dissolved inorganic carbon (DIC - CO3

2-, HCO3-, CO2), or significant

concentrations of any other potential electron acceptor.

Three springs named Grotto Pool Spring 1 (GPS1), Nipple Spring 1 (NS1) and Barnes Spring 5 (BS5) within the Cedars area were chosen as sampling sites within the area. GPS1 is a rare spring in that it allows for the collection of pristine, ultra-basic and highly-reducing water directly from the spring source. This spring possesses the highest pH, lowest Eh and the lowest cell density of the three. NS1 is characterized as having the lowest ionic concentrations and the highest methane concentrations spring. The waters from the BS5 spring, however, enter a small pool from below leading to a highly stratified water column with the bottom waters remaining highly reducing and oxygen free.

Multiple year survey of microbial community in these three springs show that the community structure is stable in anoxic water Chloroflexi spp, belonging to a new clade, dominated the Bacteria, whilst a new group likely belonging to the Euryarchaeota were the major Archaea. Eukaryotes were absent. The dominant species in the more oxic waters of the surface pools were unidentified Comamonadaceae spp similar to clones from other alkaline, non-saline environments. Three strains of Comamonadaceae spp have been isolated with optimum growth at pH 11. All grow organo-heterotrophically on a variety of substrates, while at least two strains have been shown to grow autotrophically on H2 with CO2 as the carbon source, with optimum pH at 11. Draft genomes of three isolates are available now. Combining the genomic and physiological characterization, we aim to understand how a specialized community has established and adapted to life in some of the harshest environments on Earth.

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164A In situ expression of key genes for sulfide oxidation and CO2 fixation of uncultured gigantic Aquificales bacterium dominating in hot spring microbial mats Satoshi Tamazawa*, Hideyuki Tamaki, Kazuto Takasaki, Satoshi Hanada, Yoichi Kamagata National Institute of Advanced Industrial Science and Technology (AIST), University of Tsukuba, Japan

Chemolithoautotrophic Aquificales bacteria may contribute to the sulfur cycle and the primary production in sulfidic geothermal environments such as terrestrial hot springs and deep-see hydrothermal vents, since the organisms often dominate in such environments. For instance, uncultured large sausage-shaped Aquificales (LSSA) bacterium (>90%) with 5-40 mm in cell length are predominant in white microbial mats, so-called sulfur-turf, developed in flowing sulfidic water in hot springs in Japan. Elemental sulfur particles have been observed on the cell surface of the LSSA bacterium, suggesting that the organism is likely to be involved in sulfide oxidation in the sulfidic hot springs. Very recently, through metagenomic analysis of the sulfur-turf mats, we obtained a draft genome of the uncultured LSSA bacterium and found that the organism possesses all the genes required for chemolithoautotrophic growth using sulfide as electron donor and CO2 as carbon source. To verify if the autotrophic sulfide oxidation metabolisms actually function in the sulfidic hot springs, the present study investigated in situ expression of genes associated with sulfide oxidation and CO2 fixation of the LSSA bacterium by reverse transcription (RT)-PCR analysis. Sulfur-turf mats were collected from a sulfidic hot spring of Nakabusa spa, Japan, at the sites 0, 40 and 70 cm from discharge point of the spring (50-63°C, pH 7.9-8.1), immediately immersed in RNAlater solution (Ambion), and then transferred on ice to laboratory. Total RNA was successfully extracted from the collected sulfur-turf mats by bead-beating and the RNeasy mini kit (Qiagen). Two-step RT-PCR was carried out using the designed primers specific to the genes associated with sulfur and CO2 metabolisms found in the LSSA bacterium draft genome. The RT-PCR amplified products were analyzed by agarose gel electrophoresis. As a result, the transcripts of the genes encoding sulfide dehydrogenase and sulfide-quinone reductase, which are well-known primary enzymes for sulfide oxidation, were detected at all the sampling sites. This indicates that the LSSA bacterium consistently oxidizes sulfide in the sulfidic hot spring. Further RT-PCR analysis revealed that the genes related to sulfite and thiosulfate oxidation metabolisms found in the LSSA bacterium draft genome were also expressed at all the sampling sites. Although no genes involved in elemental sulfur oxidation have been so far found in the draft genome, the LSSA bacterium may be able to oxidize sulfide to sulfate. As for CO2 fixation, the key genes encoding ATP citrate lyase, fumarate hydratase, and 2-oxoglutarate ferredoxin oxidoreductase required for reductive TCA (rTCA) cycle were also expressed at all the sites tested. This result suggests that the LSSA bacterium has an ability to fix CO2 via rTCA cycle in situ, and that the primary production in the hot springs could be supported by the organism. In conclusion, taken together with the previous findings, our study clearly demonstrated that the LSSA bacterium would grow chemolithoautotrophically using sulfide and CO2 and play a role in the reduced sulfur compounds oxidation and primary production in sulfidic geothermal environments.

164B Catabolic and stress response of Desulfitobacterium hafniense DCB-2 to increasing concentrations of 3-chloro-4-hydroxyphenylacetic acid Ye Tian*, Farai Maphosa, Maria Suarez Diez, Hauke Smidt Wageningen University, Netherlands

The extensive uses of synthetic chemicals such as organohalides have resulted in substantial waste streams and environmental pollution at the local and global scales. Many of those waste streams consist of or contain toxic chemicals which affect human as well as environmental health and ecosystem functioning when treated improperly. Therefore, we are aiming to improve the rational use of bacterial catalytic activities for the treatment, removal and prevention of organohalide pollution. To achieve the goal, we would like to understand how bacteria react to different abiotic stresses, in particular focusing on chemical stress brought about by the pollutants.

The low G+C gram positive genus Desulfitobacterium is a group of anaerobic bacteria that can use different aliphatic and aromatic organohalides as electron acceptors for their growth. Recently, the full genome sequences of Desulfitobacterium hafniense strains DCB-2 and Y51 have been elucidated, confirming their dedication to organohalide respiration as well as their versatility with respect to electron donors and alternative electron acceptors. In the present study, functional genomics and lipidomics approaches were employed to understand how D. hafniense DCB-2 cells respond to

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different concentrations of chlorophenols, using 3-chloro-4-hydroxyphenylacetic acid as a model compound.

Firstly, based on the available genome sequences, we constructed a pan-genomic microarray targeting the genomes of both D. hafniense strains DCB-2 and Y51. The array contains 21905 distinct 45–60 mer oligonucleotide probes that were designed using PICKY software. In summary, the array contains one to three probes per target transcript, and probes cover 97% of protein coding sequences and 80% of intergenic regions for both strains.

Secondly, batch cultures of D. hafniense DCB-2 were exposed to the halo-aromatic electron acceptor 3-chloro-4-hydroxyphenylacetic acid at concentrations ranging from 0 to 60mM with 20mM pyruvate as electron donor, indicating that growth was impaired from concentrations above 10mM. Maximum growth rate was observed with 10mM 3-chloro-4-hydroxyphenylacetic acid, whereas fermentative growth rate proceeded at 60% of the maximum growth rate. Growth rates in the presence of 25mM and 40mM of 3-chloro-4-hydroxyphenylacetic acid amounted to 50% and 10% of the growth rate at 10mM respectively.

Membrane lipid structure analysis revealed that cells exposed to concentrations of 3-chloro-4-hydroxyphenylacetic acid above 40mM showed an increase of the degree of fatty acid saturation. Furthermore, functional genomics approaches, including array-based transcriptomics, as well as non-gel based proteomics, were used to elucidate genome-wide responses to increasing concentrations of halogenated substrates at 10mM, 25mM and 40mM. Our results showed that 6 out of all 7 reductive dehalogenase-encoding gene clusters were up-regulated as compared to fermentative growth. In contrast, 20 sporulation related genes were down-regulated with organohalide respiration.

In conclusion, the present study provides improved insight in the activity organohalide respiring bacteria at varying pollutant concentrations, further contributing to our ability to apply these dedicated degraders in strongly polluted environments.

165A An "omics" approach to characterizing dissolved organic matter from microbially derived sources in Antarctica Michelle Tigges, Christine Foreman*, Brian Bothner, Heidi Smith Montana State University, United States

The complex mixture of organic compounds within aquatic systems, known as dissolved organic matter (DOM), comprises a significant pool of carbon on the planet. The ultimate source of DOM in many Antarctic systems is from microorganisms; however the processes leading to the release and subsequent alterations of these compounds are poorly understood. DOM is an integral component of the global carbon, nitrogen, and sulfur cycles, accounting for much of the total organic material flux in lakes, streams, and oceans. DOM also provides a carbon source for microbial activity, influencing biogeochemical and ecological processes. Despite the critical role of DOM in ecosystems, its properties and reactivity are not well defined.

The Antarctic ecosystem presents a unique opportunity for characterizing the interplay of microbial metabolism and DOM. This is due to the limited contribution to the DOM pool from plants. Characterization of DOM in aquatic environments influenced by terrestrial biomes has revealed a highly complex pool of small molecules that challenges even current high resolution chromatography and mass spectrometry equipment. In general, the complexity has limited the depth and quantification of DOM samples.

An "omics" based approach was used to chemically characterize the interaction between DOM and the representative microbial species that transform it. To do this, a comparative analysis of environmental DOM from the microbially dominated Pony Lake and Cotton Glacier aquatic systems was undertaken using advanced mass spectrometry and spectroscopy techniquesto generate an in depth molecular profile. Fundamental differences exist in the molecular profiles between the two microbially based sources of DOM. Metabolic analysis using an approach coupling ultra performance liquid chromatography to a quadrupole time of flight mass spectrometer was undertaken for cultures of representative Antarctic beta-Proteobacteria (CG3) and Flavobacteria (CG9_1) grown on a minimal media supplemented with DOM from Antarctic and terrestrial locations as a carbon source.

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Electrospray ionization mass spectrometry provided the best possible coverage of secreted metabolites and cellular metabolic composition of the organisms, thereby making available a direct indicator of cellular activity in response to the various carbon sources. Using cutting-edge analytical techniques we can follow how DOM is produced and transformed by microbes in this extreme environment.

166A Changes in the microbial water column community in response to a volcanic eruption at 9°50'N on the East Pacific Rise (EPR) Alexander H. Treusch*1, Kevin L. Vergin2, Kyle R. Pomraning2, Marie P. Johnson2, James P. Cowen3, Stephen J. Giovannoni2 1University of Southern Denmark/Nordic Center for Earth Evolution and Institute of Biology, Denmark, 2Oregon State University/Department of Microbiology, United States, 3University of Hawaii at Manoa/ Department of Oceanography, United States

Volcanic eruptions at the seafloor can significantly change the physical and chemical characteristics of the overlaying water column. Plumes form from ejected hydrothermal fluids that are highly enriched in particles and minerals when compared to the surrounding seawater. These plumes can remain stable for quite some time because of their different physical and chemical composition and are likely to harbor unique microbial communities.

After an eruptive event at 9°51’N at the East Pacific Rise (EPR) site in 2006, we used and in situ pump to retrieve three biomass samples from within plumes and one background sample from depths of 2000-2500 m. Initial analyses of the microbial community structures in the plumes using the molecular community fingerprinting method terminal restriction fragment length polymorphism (T-RFLP) showed significant differences to the background sample. Several operational taxonomic units (OTUs) were only present in the plume and not in the background sample and OTUs from the background were reduced in abundance or missing in the plume samples. To identify the OTUs that showed changes, bacterial and archaeal 16S rDNA clone libraries were constructed for all samples.

One striking finding was the high abundance of bacteria belonging to the gammaproteobacterial clade SUP05 in plume samples. They have previously been detected in plume water samples, sediments and as thiothropic endosymbionts of mussels found in hydrothermal vent ecosystems, their abundance, however, in these ecosystems has been unclear. Further interest in the SUP05 clade stems from the fact that their members are also commonly found in oxygen minimum zones in coastal marine ecosystems. The archaeal sequences identified belonged to the euryarchaeal marine group II and an uncultured group within the Halobacteriaceae as well as the crenarchaeal marine group I and the marine benthic group A.

Here we will present the results of our molecular analyses in the context of water chemistry and biogeochemistry measurements.

167A A newly isolated green alga from desert crusts - an unusual combination of a unique survival capability and record high growth and photosynthetic rates Haim Treves*, Itzhak Ohad, Aaron Kaplan Hebrew University of Jerusalem, Israel

The challenges faced by organisms inhabiting extreme niches can lead to unique properties in those that can successfully overcome them. Desert Biological Sand Crusts (BSC) represent one of the harshest environments that exist in nature. Organisms inhabiting this ecosystem must face extreme light intensities, temperature amplitude reaching from below 0oC during certain winter nights up to 60oC in day, and vast osmotic potential to changes, from close to pure rainy water to salt crystals in the crust's upper layer.

We wish to explore and identify novel mechanisms enabling a small eukaryote green alga, isolated from biological sand crusts in the NW Negev desert, to cope with a variety of stress factors that, in combination or alone, pose a survival threat to "model" algae and plants. This unusual novel organism, temporarily designated as McX, exhibits high resistance to photodamage even under extremely elevated light intensities, and survives the frequent crust hydration\desiccation cycles and vast daily temperature amplitudes.

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Taking into account the data collected on McX characteristics to date, the most intriguing one is probably its response to continuous extremely high illumination levels, varying between retaining oxygen evolution rates to even increasing them. Experiments aimed to locate a photoinhibitory light level revealed a ~30% decrease in oxygen evolution after 120 minutes of 2000 mE, but no evidence of photodamage with similar periods under lower intensities. On the other hand, exposing samples from the same McX culture to 2000 and 3000 mE (or even higher), repeatedly exhibited no decrease in oxygen evolution under the higher light level, as compared to the same above-mentioned ~30% decrease pattern of the lower one, suggesting there is a light dependant threshold for activation of a potential protection mechanism. Additional biochemical and physiological characterization of these patterns was performed yielding mechanistic insights into this organism light resistance which we wish to elaborate on. The identification of light levels inducing activation of protective mechanism may also assist in establishing detailed characterization of resistant versus vulnerable samples using proteomic and transcriptomic tools. We intend to characterizate a complete response pattern based on one or more genes or proteins providing McX with its photodamage resistance.

Nevertheless, when grown under optimal conditions, this alga, closely related to Chlorella sorokiniana, demonstrates growth and photosynthesis rates among the highest ever recorded (generation time of around 2 hours and 600-700 mmol O2 mg-1 chl h-1). We aim to unravel the mechanisms allowing McX to perform so well under laboratory conditions yet not to flourish in the sand crusts in situ. The data we intend to present suggests a highly responsive and effective carbon cycle and CCM combined with extreme morphologic changes in response to carbon source availability supporting this organism growth and photosynthetic capabilities. Following, we intend to perform a comparative transcriptome analysis of McX cultures exposed to various carbon availability treatments. In addition, genome-wide analysis of carbon cycle and CCM related genes and proteins will be held in order to identify unique molecular characteristics supporting McX performance.

167B A molecular approach to extremophilic prokaryotic biodiversity in hot springs of a little explored geothermal region in Argentina María Sofía Urbieta*1, Elena Gonzalez Toril2, Alejandra Giaveno3, Edgardo Donati1 1CINDEFI Universidad Nacional de La Plata, Argentina, 2Centro de Astrobiologia INTA CSIC, Spain, 3Universidad Nacional del Comahue, Argentina

Copahue-Caviahue region is a mountain geothermal area in the northwest of Neuquén in Argentina. The area is under the influence of Copahue volcano. The particular geological conditions generated by the presence of an active volcano favour the presence of many acidic hot springs and hydrothermal pools. The combination of low pH, high temperatures, even higher than 90° C, and high metal and sulphur concentrations make Copahue-Caviahue region an ideal environment for extremophilic microorganisms to live in. In spite of its uniqueness and potential, Copahue-Caviahue prokaryotic biodiversity has not been deeply studied yet. For this first molecular approach to the subject, water and biofilms samples from hot springs with different pH values and temperature conditions were collected. Total community genomic DNA was extracted and 16S rRNA genes were amplified with bacteria and archaea primers. Amplicons of approximately 15000 base pairs were cloned and sequenced. After sequence analysis, polygenetic trees were constructed using ARB software. Semicuantitative data was obtained by fluorescence in situ hybridization (FISH). Firstly hybridizations were done with general Bacteria and Archaea domain probes. Then, considering phylogenetic sequence information, hybridizations were done with more specific probes. Recounts were done under the epifluorescence microscope. It was possible to calculate total cells numbers for each hot spring, as well as different taxa percentages.

On the contrary of what could be expected for extreme conditions as the ones present in this acidic geothermal area, our study reveals that biodiversity was quite rich in the hot springs analyzed. We found out that archaea were the main microorganisms present when pH value was 3 or less and temperature was higher than 36°C. Moreover, when temperature was higher than 80°C 100% of the clones detected belonged to Sulfolobales order, in Crenarchaeota phylum. In hot springs where conditions were still acidic but less extreme, bacteria appear as dominant. By hybridization with specific probes we detected that Gamma and Beta-proteobacteria were the groups better represented, mainly by members of Acidithiobacillus and Thiomonas genus, respectively. Those quimioautotrophic bacteria are common in acid environments with abundant sulphur compounds. However, in Copahue-Caviahue hot springs we found as well, many heterotrophic microorganisms like Pseudomonas spp. and primary producers, like members of the Cyanobacteria phylum. In addition to well characterized

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bacteria and archaea, many sequences retrieved were similar to different uncultured clones. Those sequences are putative candidates for new species not yet isolated and characterized, and might have new metabolic characteristics that might help understand interrelations in acidic hot springs ecosystems.

In this work we aim to characterize Copahue-Caviahue prokaryotic biodiversity by non culture techniques, not only to know which microorganisms inhabit the area, but also in the hope of developing a model of its geomicrobiology. Besides, it is of great interest the possibilities of detecting, and in the future, isolating, microorganisms with particular metabolic characteristics that might be used in new biotechnological processes.

168A Response of bacterial soil communities to a small diesel-spill at the Argentine station Carlini on King George Island (Antarctica) Susana Vazquez*1, Jutta Jürgens2, Patrick Monien3, Roberto Pepino Minetti4, Walter Mac Cormack5, Helmke Elisabeth2 1University of Buenos Aires-CONICET, Argentina, 2Alfred Wegener Institute, Germany, 3University Oldenburg-ICBM, Germany, 4CIQA-UTN Regional Córdoba, Argentina, 5Argentinean Antarctic Institute, Argentina

In October 2009, a diesel pipe connecting two storage tanks at Carlini station (Antarctica) cracked, leaking and contaminating the surrounding soil which, at that time, was covered by ice and snow. Three months later, the snow began to melt and the diesel moved with runoff water through the residential and laboratory area, towards the Potter Cove. Five months after the spill, soil samples were taken at 10 sites close to, and further away, from the spill site. Marine sediment samples were also taken from Potter Cove basin, with the aim to monitor the bacterial communities in relation to the presence of hydrocarbons. One year later (January 2011), sampling was repeated at approximately the same sites and at additional ones that could not be sampled before due to the snow cover. At this time, neither smell nor visual appearance of diesel were noticeable on surface or 30 cm-depth soil, and measured total petroleum hydrocarbons decreased in the most affected sites, except in front of the pool containing the tanks where the spill originated. At this site, an underground leaking pipe had caused a more recent small event of acute contamination that is being used as a proxy to better understand the effect of the main spill on bacterial communities at Carlini station area. The impact of the spill on soils and sediments was monitored by total carbon and nitrogen content, C/N ratio, presence of hydrocarbons and differences in bacterial communities. DGGE based on the 16S rRNA gene allowed to identify the main changes in community composition over the 14-months period. Most samples showed a quite high diversity few months after the spill, as well as one year later, and only marginal changes in the taxonomic structure were observed during the study period. However, the bacterial community composition in the samples taken near the cracked underground pipe, which experienced a larger input of diesel and less time between impact and sampling, showed clear changes. The DGGE gels exhibited a reduction in diversity and an enrichment of specific bacterial groups. Clone libraries revealed enrichment in Actinobacteria and one Betaproteobacterium closely related to Polaromonas napthalenivorans, indicating an actively growing hydrocarbon-degrading community. After one more year (January 2012), there was no visual presence or smell of hydrocarbons at this site, the same that had been observed after the main spill. Our results show that in Potter peninsula the bacterial communities undergo a fast shift to lower diversity and higher dominance when a sudden spill occurs on a soil with certain history of exposure to hydrocarbons. The unusually fast disappearance of the spilled diesel in such a cold habitat is favoured by the kind of diesel used (containing mostly light aliphatic hydrocarbons), the sandy texture of soil, snowmelt runoff, summer rains and a well adapted microbiota, with a promising oil-bioremediation potential.

169A Enzymes from extremophilic microorganisms; tools to reduce energy consumption in industrial processes Jan Vester*, Mikkel Glaring, Peter Stougaard University of Copenhagen / Department of Plant and Environmental Sciences, Denmark

When enzymes are used in industrial processes, heating is often required in order to reach a temperature where the enzymes are active, and thus fossil fuels are consumed. These enzymes are typically obtained from mesophilic microorganisms, and these enzymes are not efficient or functional when the temperature is lowered. Microorganisms living in a cold environment, however, have

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enzymes adapted to function at low temperatures. These enzymes can potentially replace existing mesophilic enzymes, currently used in industrial processes. This could reduce energy consumption as well as problems with contaminations resulting in spoilage (e.g. in food production).

Ikaite tufa columns from Southwest Greenland constitute the only known alkaline (pH 10.5) and cold (2-6 °C) environment on Earth. The columns harbour a variety of Gram positive and Gram negative bacteria. Analyses of strain collections and metagenomic sequencing show that ~30% of the isolates represent new species and genera and that the majority remains uncultured.

Enzymes from cultured ikaite-bacteria have been documented to be cold-active. To explore the enormous potential of the so far uncultured microorganisms, we present a metagenomic approach currently being conducted in order to identify new enzymes independent of cultivability.

A functional metagenomic expression library has been established and is currently being screened for enzymatic activities of industrial relevance that are cold active and/or active at alkaline conditions. The library has been constructed using a vector which allows for expression in different Gram negative hosts, increasing the chance of identifying relevant enzymatic activities. Once activities are identified, genes encoding the enzymes will be used for recombinant expression followed by characterisation of the enzymes.

Some of the total diversity is lost when preparing the DNA for the functional expression library. This decrease in diversity has been analysed by pyrosequencing of the 16S gene. This has shown that the phyla dominant in the starting material (Proteobacteria and Firmicutes) are also dominant in the library, whereas several of the less abundant phyla (e.g. Bacteroidites and Cyanobacteria) are almost completely lost.

The potential of hitherto uncultured microorganisms is enormous. This study aims at utilising some of this potential to identify new enzymes with characteristics that are of interest to industry and potentially could reduce energy consumption and/or improve product quality.

170A Reconstruction of the microbial methane cycling in deep Arctic environments Dirk Wagner*1, Juliane Bischoff2, Kai Mangelsdorf1 1Helmholtz Centre Potsdam, GFZ German Centre for Geosciences, Germany, 2Alfred Wegener Institute for Polar and Marine Research, Germany

Microbiological studies over the last two decades have shown the existence of diverse and active microbial ecosystems in the deep subsurface. It has been estimated that out of the total pool of prokaryotes inhabiting the Earth, between 75% and 94% occur in deeply buried marine and terrestrial sediments. Another surprising fact is that the biomass of the deep subsurface ecosystems is greater in numbers than the one in surface near habitats. This indicates the fundamental role of biomass from the deep biosphere for the global biogeochemical cycles over short and long time scales.

However, the deep subsurface of the Earth, especially in arctic environments, remains relatively unexplored in the field of microbiology. Deep sediments of the Arctic play a key role for the Earths’ climate because of the huge amounts of belowground carbon that are preserved in the frozen ground and the fact that global warming is most pronounced in polar regions. Especially the thawing of terrestrial permafrost is suggested to be associated with a massive release of greenhouse gases, in particular methane. To understand how the system will respond to climate changes it is not only important to investigate the current status of microbial carbon turnover but also to reconstruct the systems’ response to climate changes in the past. Therefore, a comprehensive study was conducted, comprising 400 ka old archives of past microbial activity and recently active microorganisms in terrestrial permafrost deposits recovered from central Lena Delta and lake sediments in Chukotka, northeast Siberia.

Using a broad set of analytical methods (including ribosomal RNA gene based approach and lipid biomarker analyses) in Middle to Late Pleistocene deposits we show a strong correlation between organic matter concentration and microorganism abundance. Lipid biomarkers that are stable in geological time scales were used to reconstruct the past microbial communities and their response to

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climate changes. In particular, archaeol demonstrated changes in the abundance of methanogenic archaea throughout the last 42 ka. This suggests, past warming trends caused an increase of methanogenic communities, while cooling trends cause a decline.

Furthermore, analyses of phospholipid esters (PLFAs) and ethers (PLELs), characteristic markers for living bacteria and archaea, suggest the presence of living microbial cells in up to 400 ka old deposits. This was supported with incubation experiments, where significant methane production rates were observed.

Our results show a quantitative and qualitative temperature response of the microbial communities in deep arctic deposits to past climate changes. Microorganisms do not only survive in the frozen ground, but they can be also metabolic active under energy limited conditions contributing to the carbon transformation in arctic environments.

171A New nitrifying microorganisms from hot springs of Yellowstone National Park Katja Wendt*, Yvonne Bedarf, Eva Spieck Universität Hamburg, Germany

Nitrification plays a significant role in the global nitrogen cycle, involving the microbial oxidation of ammonia via nitrite to nitrate. Two physiological groups of chemolithoautotrophic microorganisms, which perform this process, are referred to as ammonia-oxidizing bacteria or archaea and nitrite-oxidizing bacteria. Until now, the only cultured thermophilic ammonia oxidizing representative, growing at 72°C, is the archaeon 'Candidatus Nitrosocaldus yellowstonii'. Known thermophilic nitrite oxidizers belong exclusively to the genus Nitrospira, but the identity of nitrite oxidizers growing above 60°C is still unknown. Nitrification takes place in a variety of moderate and extreme environments, including hot springs and mud pots. Biofilms and sediments from two springs in Yellowstone National Park (USA) were used as sample material for the selective enrichment of thermophilic ammonia- and nitrite oxidizers.

Growth curves, performed with different enrichments at 70°C, confirmed the successful cultivation of ammonia oxidizers through the repeated conversion of ammonia to nitrite. Light, fluorescence and transmission electron microscopic investigations revealed the existence of coccoid cells as well as rods in these cultures and rods could be affiliated to the domain Bacteria by FISH analysis.

Analyses of the 16S rRNA revealed the occurrence of Nitrosocaldus yellowstonii in the initial enrichment culture as well as other nitrifying Thaumarchaeota (85-98% similarity). In accordance, the archaeal ammonia monooxygenase gene (amoA), a subunit of the key enzyme of aerobic ammonia oxidation, was detected. However, a community shift occurred by transferring the culture and no more archaeal genes (16S and amoA) could be detected any more although ammonia oxidation continued. Clone analyses of this later enrichment gave hints for the presence of Thermomicrobium roseum (98-99%), Ammonifex degensii (84%) relatives of Sphaerobacter thermophilus (82-88%) and Thermobaculum terrenum (82%). Immunofluorescence labeling of rods and filaments with polyclonal antibodies targeting the bacterial AmoB indicated that ammonia oxidizing bacteria appeared in the enrichment culture, but they still have to be correlated with a 16S rRNA sequence.

Next to the consumption of ammonia, the growth curves of two ammonia-oxidizing cultures presented a simultaneously turnover of nitrite to nitrate and thus the existence of co-cultured nitrite oxidizers. From these cultures, we successfully enriched microorganisms in mineral medium with nitrite as sole energy source. These cultures are dominated by short rods, which despite successful experiments do neither react with the general bacterial (Eub 338I) nor the archaeal (Arch 915) oligonucleotide probes. Furthermore, flexible rods could be observed, which were identified as Thermoleophilum album by 16S rRNA analysis (99%). Short rods grow at 65°C and differ obviously from Nitrospira in cell shape, but their identity remains unknown until now.

Currently, both ammonia and nitrite oxidizing enrichments originating from Yellowstone Nation Park grow very slowly at 65°C. Nevertheless, with polyphasic approaches it has succeeded to get a first insight into the enlarged diversity of thermophilic nitrification, which existence was indicated at temperatures up to 97°C by in-situ measurements. Especially, it remains interesting to clarify the identity of thermophilic nitrite oxidizers, the rather neglected second group of nitrification.

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172A Microbial biodiversity of the Alpine vanishing cryosphere Linda Wilhelm*, Katharina Besemer, Christina Fasching, Gabriel Singer, Tom J. Battin University of Vienna, Department of Limnology, Austria

Glacial retreat occurs at unprecedented pace worldwide. Despite this rapid retreat, many mountain glaciers still nurture headwaters, the smallest in alpine fluvial networks. In these streams, glacial runoff dynamics is known as a major control of algal and macroinvertebrate ecology. While glaciers are increasingly recognized to harbor active microbial communities, possible implications of glacial retreat for the microbial ecology of glacial streams and particularly for their biofilms — the major players in stream processes — remain elusive. Here we provide a first systematic survey on microbial community structure and biodiversity, based on massive 454-pyrosequencing of the 16S rRNA gene, in the glacial ice, runoff and in stream benthic biofilms at the terminus of 26 Alpine glaciers. Our results suggest strong differences in microbial diversity and community composition in the ice and both stream habitats. Streamwater microbial diversity significantly decreased with elevation, whereas biofilms did not. Environmental parameters, such as streamwater conductivity, pH and temperature, explained much of the variation in community composition in biofilms, while space and sampling time were insignificant. Conductivity and pH, but not temperature, even influenced the relative occurrence of certain bacterial phyla (for example Nitrospira) in biofilms. Our results underscore the sensitivity of microbial communities to environmental change as glacial retreat is induced. This adds a new dimension to our understanding of glacial ecosystems as part of the vanishing cryosphere.

173A Response of bacterial and archaeal ammonia oxidizers to abruptly increased moisture in the soil of arid ecosystems: abundance of amoA genes and transcripts Sher Yonatan, Ronen Zeev*, Nejidat Ali Ben Gurion University of the Negev, Israel

In desert ecosystems, the rates of biogeochemical cycles are primarily regulated by rain events and are spatially associated with soils under the canopy of perennial shrubs. During the dry period, soil microorganisms survive by accumulating compatible osmolytes within the cytoplasm. Upon the first rain event and the sudden increase in water activity a significant fraction of the microbial biomass can be lost due to osmotic shock. Consequently, the released nutrients have the potential to trigger increased rates of activities and nutrient cycling. Nitrogen availability is generally considered to be a limiting factor for primary production in pristine desert ecosystems. Nitrification, particularly during the wet period, is detrimental to preserve the nitrogen pools in the ecosystem. Nitrate is easily leached below the roots zoon and is prone to denitrification activity (reduction of nitrate to atmospheric nitrogen). Both bacterial and archaeal species have the potential to oxidize ammonia to nitrite (first step of nitrification). Therefore, the aim of this study was to determine the survival (abundance and activities) of both ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) after an abrupt moisture increase in a dry desert soil. It is hypothesized that AOB and AOA will exhibit differential responses. The abundances of the bacterial and archaeal amoA genes (codes for sub-unit A of ammonia monoogygenase) and their respective RNA transcripts were determined before and after the first substantial rain event after a prolonged dry summer.

Soil samples were collected from Avdat LTER, a hyper arid area in the Negev desert, Israel. Samples were taken from under the canopy of Zygophyllum dumosum shrubs (UC) and inter-shrub patches (ISP) at the end of the dry season (October 9, 2011) and immediately after the first substantial rain event (5mm/day) (January 20, 2012). Nucleic acids were extracted and RNA was reverse transcribed with a bacterial amoA forward primer and an archaeal amoA reverse primer. Archaeal and bacterial amoA gene and gene transcripts were quantified by qPCR.

Soil salinity, nitrate concentrations and the abundance of the bacterial amoA gene decreased significantly immediately after the rain event, while the archaeal amoA abundance did not change significantly. Subsequently, ratios of archaeal amoA/bacterial amoA gene copies increased after the rain event. Ratios of the copies in soils UC increased after the rain event, from 78 to 282. In the ISP, bacterial amoA gene transcripts/gene ratios were in the range of 500 and were not affected by the rain event. Ratios of archaeal amoA transcripts/gene were substantially low and were not affected by the rain event or by patch type (UC vs. ISP).

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These results suggest that AOA are more resistant to a prompt increase in soil moisture content. Furthermore, the higher ratios of the bacterial amoA gene transcripts/gene in the UC soil after the rain event indicate a fast response of AOB to increasing ammonia availability, which can eventually contribute to nitrogen losses from the ecosystem.

174A Characterization of H2-producing CO-oxidizing bacterium Carboxydothermus pertinax from an acidic hot spring in Japan Yasuko Yoneda*, Takashi Yoshida, Satoshi Kawaichi, Takashi Daifuku, Takayuki Kitamura, Takahiro Inoue, Sanae Kano, Keiji Takabe, Yoshihiko Sako Kyoto University, Japan

Carbon monoxide (CO) is the common trace gas in hydrothermal environments. Hydrogenogenic CO-oxidizers have been isolated from these environments, and their ecological role is presumed as important "CO-scavengers" for CO vulnerable co-habitors. Isolation source of them had been limited to around neutral pH (5.5-8.4). We recently isolated H2-producing CO-oxidizing bacterium, Carboxydothermus pertinax Ug1T from an acidic hot spring (pH 2.7). Here we report isolation and characterization of this new isolate.

Sample of hot spring was collected from Unagi-onsen, located in Kagoshima Prefecture, Japan. The sample temperature was approximately 50°C, and the pH was 2.7. B medium (pH 6.2) was prepared and dispensed in glass test tube. Medium B contained: 30 mg Na2SiO3, 1.0 g NH4Cl, 0.50 g KH2PO4, 0.30 g MgSO4･7H2O, 0.50 g CaCl2･2H2O, 0.30 g KCl, 0.10 g NaCl, 0.10 g NaHCO3, 0.50 mg resazurin, 1.0 ml trace mineral solution SL 6, 1.0 ml vitamin solution, and 0.10 g yeast extract. Each glass tube was flushed with pure CO gas and sealed immediately. Sample was inoculated to medium using syringes. The purity of an isolate was checked routinely by microscopic observation and sequencing of partial 16S rRNA gene. CO and H2 concentration in head space was analyzed using GC-2014 (Shimadzu). Fe(III) oxidation was detected as Fe2+ concentration in liquid phase using spectrophotometerical ferrozine method. Phylogenetic analysis of new isolated was conducted based on 16S rRNA gene.

After incubation at 55°C for 2 days, cell growth with H2-production and CO consumption was observed. Culture was transferred to fresh modified B medium supplemented with ferric citrate (Bfc medium) and incubated at 65°C. After a 10-fold dilution to extinction using Bfc medium, we established a new bacterial strain Ug1T. The growth condition was as follows: 50-70°C (optimum at 65°C), pH 4.6-8.6 (optimum at pH 6.0-6.5). Cell of the new isolate was motile and rod-shaped (1.0-3.0 µm in length). Four copies of 16S rRNA gene were identified from the new isolate. The 16S rRNA gene sequences were most similar to the members of the genus Carboxydothermus (94.1-96.6%). Phylogenetic tree using 16S rRNA genes of Carboxydothermus and Moorella was constructed. 16S rRNA genes of strain Ug1T formed an independent clade to the other Carboxydothermus members indicating new isolate as a novel species. The isolate produced H2 and CO2 during growth on CO. Reduction of ferric iron also occurred simultaneously. The isolated was capable of reducing elemental sulfur or thiosulfate during CO-oxidation. These physiological features had not been described in the other Carboxydothermus members.

Based on distinct phylogeny and physiology, strain Ug1T as a novel species of Carboxydothermus, with name Carboxydothermus pertinax sp. nov., is proposed.

175A Ammonium removal by sulfate- reducing and nitrate- dependent anammox Ivar Zekker*, Ergo Rikmann, Toomas Tenno, Priit Vabamäe, Kristel Kroon, Alar Saluste, Taavo Tenno University of Tartu, Estonia

Treatment of N-rich waste flows has high costs on external carbon source, aeration and utilization of excess sludge when nitrification-denitrification methods are applied. The nitrite-dependent anaerobic ammonium oxidation (Anammox) process has been proven to be feasible for biological nitrogen removal from nitrogen-rich waste streams (Strous et al., 1999).

SO42- reduction by Anammox bacteria has been experimentally observed (Fdz.-Polanco et al. 2001). A

Planctomycetes bacterium Anammoxoglobus sulfate, capable of oxidizing NH4+ into NO2

- using SO42-

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as an electron acceptor, was isolated by (Liu et al., 2008). Fdz.-Polanco et al. (2001) proposed a summary equation describing the two-staged sulfate-reducing ammonium oxidation (SRAO) process, which has later been complemented with a possibility for sulfide formation noted by Lei et al. (2009).

Fdz.-Polanco et al. (2001) studied anaerobic treatment of vinasse wastewater (with high COD, total Kjeldal nitrogen and sulfate concentrations of 27 g/L, 2.3 g/L and 1 g/L, respectively), observing significant removal of nitrogen (up to 55%) and sulfate (up to 99%), with low sulfide generation. Alternative electron acceptors such as SO4

2- may provide opportunities to reduce the need for aeration in the nitritation step preceding the Anammox process.

In this study, we aimed to find out the feasibility of SRAO process in case of removal of ammonium from reject water, and compare the efficiency and stability of the process with the nitrite-dependent Anammox in upflow anaerobic sludge blanket reactors (UASBRs).

A 0.75 L thermostated UASBR1 (for SRAO) and a 1.5 L volume UASBR2 (for nitrite-dependent Anammox) with internal recirculation were operated in parallel. The influent (diluted rejection water+synthetically added nitrite or sulfate) was fed by periodically switched-on peristaltic pumps (SEKO, Italy).

The UASBRs were seeded with anaerobic sludge having the potential for carrying out the Anammox process (containing unclassified Planctomycetaceae uncultured bacterium). It was obtained from the facility treating sulfate-rich wastewater of the Salutaguse yeast factory (Salutaguse, Estonia).

SRAO and nitrite-dependent Anammox process were rapidly started up for the treatment of supernatant from sludge digestion, although SRAO was much less efficient than the "conventional" Anammox process despite higher temperature (36 (±0.5)°C) applied. Used inoculation sludge showed immediate adaptation (within a week) to different composition wastewater's.

According to the one-way ANOVA analysis of variance nitrite-dependent Anammox process showed significantly higher TN removal rates than sulfate-reducing Anammox process. Both processes start-up for treatment of reject water was not successful, as SRAO process did not show further improvement in adapting to increased influent loading rates as compared with nitrite-dependent Anammox process. For the UASBR1 increasing NH4

+ loading rate 60% in the period II had no significant effect on TN removal rate. According to (Fdz Polanco et al., 2001), the SRAO process being applied for treatment of wastewater containing high concentrations of nitrogen, sulfate and easily biodegradable organics resulted in TN removal rates of 0.13 kg-N/(m³ day).

131B Characteristics of microbial community structure during desertification reverse procedure Wei Zhang*, Gaosen Zhang, Guangxiu Liu Cold and Arid Regions Environmental and Engineering Research Institute, Chinese Academy of Sciences, China

Desertification is a common environmental challenge for humans. Biotechnologies have been used for the management of soils in areas of desertification and the use of microorganisms has shown potential benefits, but such research is still in the initial stages. During our recently researches, we have studied the characteristics of microbial community structure in soil crust and the soil beneath the crust along sand dunes of different fixed ages at the southeast edge of the Tengger Desert, China, using culture dependence and independence methods. The Shapotou and Yiwanquan regions were selected as sampling sites, which represented an artificial and a natural restoration region, respectively. Results showed that the number of culturable bacteria increased with the degree of sand dune fixation, and decreased with depth, which was similar to the trend of soil improvement in areas of desertification. Such characteristics did not show any significant differences between the artificial restoration region and the natural restoration region (P>0.05). The genus Arthrobacter and Bacillus were the predominant culturable bacteria at the southeast edge of the Tengger Desert, which increased and decreased with the degree of sand dune fixation respectively. This result implied that Arthrobacter may play a significant role during preliminary soil crust formation. The results also showed that the quantity of culturable bacteria was positively correlated with the concentration of total soil carbon and total soil nitrogen (P<0.05), but negatively correlated with pH values (P<0.05). These results showed that

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microorganism number was closely related with the improvement status of desertification. Based on 16S rDNA gene sequences and the phylogenetic tree, the culturable bacteria in the study area belonged to 18 genera and fell into six phylogenetic groups: Acidobacteria, Firmicutes, α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria and Bacterioidetes. Blast results also showed that these bacterial strains had a variety of functions. Based on culture independence method, phylogenetic analysis of DGGE map revealed the presence of a rich diversity of bacteria, including Proteobacteria, Acidobacteria, Bacterioidetes, Actinobacteria, Firmicutes, Chloroflexi, Nitrospirae and Cyanobacteria. The Proteobacteria, consisting of the α, βand γsubdivisions, were clearly the dominant group at all depths. The bacterial community abundance closely correlated with soil enzyme activities in each of the different soils. The presence of Cyanobacteria correlated with significant increases in protease, catalase and sucrase in the soil crust, and increased urease in the rhizosphere soil of Artemisia ordosica. The occurrence of Acidobacteria was associated with significant increases in urease, dehydrogenase, sucrase in the rhizosphere soil of Caragana korshinski. The presence of γ-Proteobacteria correlated with a significant increase in polyphenol oxidase in the rhizosphere rhizosphere soil of Artemisia ordosica. The study indicated a close relationship between the soil bacterial community and soil enzymes, suggesting further investigations into bacterial function in this desert ecosystem.

176A Ribosomal tag pyrosequencing reveals that sulfur metabolizing microbes drive the shallow-sea hydrothermal systems off NE Taiwan's coast Yao Zhang*1, Zihao Zhao1, Chen-Tung Arthur Chen2, Nianzhi Jiao1 1Xiamen University, China, 2National Sun Yat-Sen University, Taiwan

To determine microbial community composition, community spatial structure and possible key microbial processes in the shallow-sea hydrothermal vent systems off NE Taiwan's coast, we examined the bacterial and archaeal communities of four samples collected from the water column extending over a redoxocline gradient of a yellow and four from a white hydrothermal vent. Ribosomal tag libraries based on DNA and RNA showed statistically significant differences between the bacterial and archaeal communities of the different hydrothermal plumes. The bacterial and archaeal communities from the white hydrothermal plume were dominated by sulfur-reducing Nautilia and Thermococcus, whereas the yellow hydrothermal plume and the surface water were dominated by sulfide-oxidizing Thiomicrospira and Euryarchaeota Marine Group II, respectively. Canonical correspondence analyses indicate that methane (CH4) concentration was the only statistically significant variable that explains all community cluster patterns. However, the results of pyrosequencing showed an essential absence of methanogens and methanotrophs at the two vent fields, suggesting that CH4 was less tied to microbial processes in this shallow-sea hydrothermal system. We speculated that mixing between hydrothermal fluids and the sea or meteoric water leads to distinctly different CH4 concentrations and redox niches between the yellow and white vents, consequently influencing the distribution patterns of the free-living Bacteria and Archaea. We concluded that sulfur-reducing and sulfide-oxidizing chemolithoautotrophs accounted for most of the primary biomass synthesis and that microbial sulfur metabolism fueled energy flow and element cycling in the shallow hydrothermal systems off the coast of NE Taiwan.

177A Could organic compounds in a storm cloud support the diverse bacterial community revealed by hailstones? Tina Šantl Temkiv*1, Kai Finster1, Thorsten Dittmar2, Bjarne M. Hansen1, Runar Thyrhaug3, Niels W. Nielsen4, Ulrich G. Karlson5 1Aarhus University, Department of Bioscience, Denmark, 2University of Oldenburg, Institute for Chemistry and Biology of the Marine Environment, Max Planck Research Group for Marine Geochemistry, Germany, 3University of Bergen, Department of Biology, Norway, 4Danish Meteorological Institute, Denmark, 5Aarhus University, Department of Environmental Science, Denmark

Cloud droplets are inhabited by diverse bacterial communities that may have important impacts on physical and chemical processes in the atmosphere. Due to their inaccessibility and extremely short lifetimes, storm clouds are among the least studied habitats of the atmosphere. During their formation inside storm clouds, large hailstones collect >109 supercooled cloud droplets. We have carried out the first comparative study, analysing 50 individual large hailstones as replicate samples, in order to study the dissolved organic matter as well as the microbiome of a storm cloud.

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We found that the concentrations of total dissolved nitrogen (30 µM, Q1–Q3 = 27–35 µM) and dissolved organic carbon (Me=179 µM, Q1–Q3 = 132–220 µM) were both high. Ultrahigh-resolution mass spectrometry was used to analyze the high molecular mass range of dissolved organic matter, by which we identified molecular formulae of almost 3000 compounds. The majority of compounds (60%) were highly unsaturated or phenolic organic acids, typical for soil-derived dissolved organic matter. Less than 3% of the identified compounds were plant waxes, fatty acids or carbohydrates, representing the only group of compounds structurally suitable for microbial degradation. Based on the average bacterial densities (Me=1973 cells/ml, Q1-Q3=1485-2960) and assumed initial cloud droplet diameter of 10 µm, we estimated that only 1 out of 106 cloud droplets carried a bacterial cell. Cloud water thus proved to be a sparsely populated and nutrient-rich microbial environment, where significant bacterial growth is feasible even if only 3 % of dissolved organic matter is usable as bacterial substrate. As the residence time of bacteria in cloud droplets is short, only bacteria with opportunistic ecological strategy, that is having fast growth responses and fast growth rates are likely to grow in clouds.

We obtained DNA clone libraries with a total of 485 gene sequences, demonstrating a diverse bacterial community with representatives from 11 phyla. The bacterial community had a medium species evenness and a high species richness, with approximately 1800 OTUsat the species level estimated for the whole cloud. We isolated 424 bacterial strains, which belonged to 23 genera. Approximately 60% of those were Gram-negative, mostly belonging to plant-associated genera Methylobacterium and Bradyrhizobium. Methylobacterium spp. are known to carry several rRNA operons, facilitating fast growth responses and high growth rates. In addition, analyzing their metabolic potential to degrade 95 organic compounds with Phenotype Microarray plates, we found that they were generalists, growing on many diverse organics. These both indicate their opportunistic ecologic strategy, making them a candidate for active growth in the clouds. In addition, they are likely well adapted for survival in the atmosphere, as similar stress factors, for example UV radiation and desiccation, are shared between plant surfaces and the atmosphere. Based on the spectrum of bioavailable organics as well as bacterial numbers and types, we conclude that storm clouds likely contain growing bacteria.

178A Investigation of low bacterial density environments: Reducing background contamination problems with sensitive DNA-targeting techniques Tina Šantl Temkiv*, Kai Finster, Ulrich G. Karlson Aarhus University, Denmark

Microbial life in some extreme environments may be so scarce that its detection requires extremely sensitive DNA-targeting techniques, which are compromised by the ubiquitous presence of contaminant DNA. Studying bacterial diversity of large hailstones, which are extreme environments characterised by the presence of very few bacterial cells, we show that a simple DNA-extraction method together with an efficient decontamination procedure can reduce the background contamination. Due to the combination of low bacterial density and low volume of our samples, a semi-nested PCR, which can amplify even just a few DNA molecules, was required for DNA amplification prior to the construction of clone libraries. To extract DNA from the hailstone samples, we used a very simple, one-step DNA extraction procedure, which reduced the loss of indigenous DNA as well as the amount of contaminant DNA introduced. In addition, a modified multistrategy DNA decontamination procedure, which was previously developed for working with ancient DNA, was carried out on laboratory surfaces, gloves, labware and reagents used in the first PCR reaction, in order to either eliminate or at least reduce the amount of contaminating DNA. We confirmed that the decontamination technique was effective by analysing the bacterial diversity of 9 hailstones, and contrasting it to the diversity found in 9 negative controls, which indicated the amount and the type of the background contamination. The obtained clone sequences from the negative controls (N=295) and the hailstone samples (N=485) confirmed that the samples were much more diverse and that the diversity of the background contamination was final and could be well described. In addition, the background contamination sequences were classified to only 9 known genera, whereas the hailstone sequences were affiliated to as many as 55 genera. In conclusion, we show that such a decontamination procedure can be efficiently employed for the study of low bacterial density environments in order to prevent false positive results. The analysis of several negative controls along with the samples can ensure the reliability of results, even in cases where contamination cannot be entirely eliminated.

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179B Atmospheric methane oxidation by airborne methanotrophic bacteria at cloud-like conditions Tina Šantl Temkiv*1, Kai Finster1, Bjarne M. Hansen1, Lejla Pašić2, Ulrich G. Karlson1 1Aarhus University, Denmark, 2University of Ljubljana, Slovenia

The dominant sink of atmospheric methane is photooxydation by hydroxyl radicals, but methanotrophic bacteria in top soil layers are considered to account for 5% of its sink. Top soils are a prominent source of airborne bacteria, which degrade some organic atmospheric compounds at rates similar to photooxydation. Although airborne methanotrophs would have direct access to atmospheric methane, which is also the most abundant organic compound in the atmosphere, their presence and activity in the atmosphere has so far not been investigated. By enriching airborne methanotrophs from air and rainwater on 2% methane, we confirmed their presence in the atmosphere. Besides growing on methane at high concentrations, the enrichment contained high affinity methanotrophs that could oxidize methane at atmospheric concentrations. Clone libraries of 158 pmoA gene sequences revealed the presence of seven OTUs, which were affiliated to type I methanotrophic genus Methylocaldum and type II genera Methylocystis and Methylosinus. Isolates of Methylocystis were shown by earlier studies to exhibit high-affinity methane oxidation. By constructing clone libraries of 198 16S rRNA gene sequences, we found that enrichments also contained members of non-methanotrophic genera Hyphomicrobium, Variovorax and Pseudomonas, previously found in high affinity methanotrophs enrichments. We investigated the effect of simulated cloud-like conditions, i.e. the acidic medium and different concentrations of common cloudborne organic compounds, on methane oxidation. Although both low pH and the presence of cloudborne organics (acetate, formate, succinate, and oxalate) negatively affected methane oxidation, airborne methanotrophs could degrade atmospheric methane in most cases. The discovery of cultivable airborne methanotrophs expands our knowledge on the global distribution of methanotrophs and may also change our current understanding of atmospheric methane turnover, as airborne bacteria may serve as an additional sink of atmospheric methane.

180A Elevated CO2 is changing soil microbial communities at natural CO2 springs (mofettes) Nataša Šibanc1, Thorunn Helgason2, Alex J. Dumbrell3, Ines Mandič Mulec4, Polona Zalar5, Hans-Josef Schroers6, Irena Maček*1 1University of Ljubljana, Biotechnical Faculty, Department of Agronomy, Slovenia, 2University of York, Department of Biology, United Kingdom, 3University of Essex, School of Biological Sciences, United Kingdom, 4University of Ljubljana, Biotechnical Faculty, Department of Food Science and Technology, Slovenia, 5University of Ljubljana, Biotechnical Faculty, Department of Biology, Slovenia, 6Agricultural Institute of Slovenia, Slovenia

Natural CO2 springs (mofettes) are specific and extreme ecosystems, characterized by high soil CO2 and reduced soil O2 concentrations, leading to a locally hypoxic environment. Despite decades of research into different aspects of plant biology in mofette areas, research into soil microbial diversity has so far been minimal and thus represents an outstanding and important area of research. This is the first in depth study of soil biodiversity from mofette sites, and includes quantifying fungi (arbuscular mycorrhizal fungi, yeasts, other soil fungi), soil bacteria, and archaea. Soil and plant roots were sampled from areas with a long-term extreme exposure to geological CO2 (up to 99.9%). For molecular characterisation of soil microorganisms several methods were used, including DNA extraction, polymerase chain reaction, cloning, terminal restriction fragment length polymorphism, sequencing and massively parallel pyrosequencing. In addition to molecular identification, taxonomic and biochemical approaches were used to identify non-mycorrhizal fungi. Our results revealed significant turnover in soil microbial communities between sites that are highly exposed to geological CO2 gas emissions compared to control sites across all microbial groups that were included in our study. We have shown that some arbuscular mycorrhizal fungi are more strongly associated with local variations in the soil environment, particularly hypoxia, than with the distribution of their host plants. On the basis of this research we suggest that assemblages of arbuscular mycorrhizal fungi are the result of powerful selection by, and local adaptation to, the soil environment. In addition to these findings a new species of yeast related to Occultifur extrenus was indentified and several other fungal species were isolated from the mofette soils, including five Penicillium isolates that may represent previously undescribed species and are currently being tested for identification. A big shift in communities of soil bacteria and archaea between control sites and sites exposed to geological gases has also been observed within this study. In addition to giving a new insight into this natural phenomena, our work also has a potentially high impact application to a wider stakeholder community; natural CO2 springs

Thursday 23 August


could serve as model systems that enable the investigation of the potential risks to naive ecosystems of leakage of CO2 from long-term geological storage of carbon in carbon capture and storage systems.

thursday 23 august ps24 – microbial life in extreme ...llebbe/abstracts/isme 14... · in order to...

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