monday 20 august ps03 – microbes in a changing oceanllebbe/abstracts/isme 14... · ps03 –...

Monday 20 August

1 PS03 – Microbes in a Changing Ocean

PS03 – Microbes in a Changing Ocean

297A Health aspects of dry sand of the beaches in the state of Paraná-Brazil in terms of mechanical revolving Sumaia Andraus*1, Jair Alves Dionisio2, Ida Chapaval Pimentel2 1Instituto Ambiental do Paraná, Brazil, 2Universidade Federal do Paraná, Brazil

The level of microbiological contamination in the beach sand is typically higher than the contamination observed in water, that is because of the phenomenon of bioaccumulation, tide conditions, rain intensity, presence of drains, among other situations. The survival of bacteria in the sands of the beaches is affected by a number of factors, organic matter, moisture, temperature, sunlight, water holding capacity, pH, among others. The research was developed to evaluate the efficiency of the dry sands of mechanical tillage in reducing the bacteria indicator of fecal contamination (BIFC). In summer 2011/2012 collections were made of dry sand on the beaches of Matinhos (MS-1 and MS-2), Caiobá (CA-1 and CA-2) and Guaratuba (GA-10 and GA-14), local georeferenced and that part of the monitoring network designed by bathing the Environmental Institute of Paraná. The Sanitation Company of the State of Paraná-Sanepar held the revolving mechanical diary of dry sand, with removal of garbage, run by machines coupled mats. Seven samples were taken, three on dec./11, two on jan./12 and two on feb./12, the first being collected prior to tillage. In an area of 50 m2 (10 m x 5 m) were collected composite samples of ten sub-samples, obtained from the 0-10 cm. Density (NMP/200 g sand) from Escherichia coli (EC) was determined by enzymatic method cromofluorogenic using broth culture medium Fluorocult-LMX. On the beach of Matinhos, the density of E. coli was reduced from 13.8 to 84.6%, in Caiobá beach, CA-1 point, there was a reduction of 7.8 to 19.6%, while in point CA-2 had an increase of 200% to 390%, and the beach at the point Guaratuba GA-10 showed an increase from 225 to 2300%, with the exception of the last collection, which reduced to 40%, while in GA-14 point decreased from 3.7 to 67.3%. The revolving contributed to decreased bioaccumulation, thereby reducing the density of BIFC, especially when the rainfall and the population not interfere. It was also found, a visual enhancement on the physical aspect of the sands, on the basis of trash removal and revolving.

298A Microbial buffering: protecting The Great Barrier Reef against anthropogenic impacts Florent Angly*1, Candice Heath1, Matthew Sullivan2, Virginia Rich2, Britta Schaffelke3, David Bourne3, Gene Tyson1 1Australian Centre for Ecogenomics, Australia, 2University of Arizona, USA, 3Australian Institute for Marine Science, Australia

Coral reefs are productive and diverse ecosystems but are declining worldwide due to human activities such as fishing and agriculture. The Great Barrier Reef, an iconic Australian resource, is protected from fishing but is susceptible to the agricultural fertilisers and pesticides found in runoff waters. Microorganisms are an essential part of coral reef ecosystems, encode a large array of enzymes and have rapid generation times. We hypothesise that microorganisms can degrade pesticides and recycle nutrients found in land-based anthropogenic runoff, thus protecting the Great Barrier Reef against human-induced stresses. During 3 years, we collected water samples in the Great Barrier Reef lagoon along a 120 km transect exposed to a gradient of runoff water and for which extensive water quality data exists. The microbial samples were characterised using 16S rRNA amplicon pyrosequencing. The profiling of microbial communities revealed that the microbial composition changed significantly between the dry season and the wet season (during which runoff is more abundant). We also found that microbial communities close to the runoff source were enriched in Thermoplasmatales, a group of Archaea previously found in association with corals. At the same site, a likely bloom of Oleibacter, a newly described microorganism that metabolises hydrocarbons, was also observed after the occurrence of a cyclone. These results suggest that specific microorganisms respond to runoff and effectively buffer the reef from anthropogenic activities. Phages, viruses of bacteria, may promote a quick adaptation of these microorganisms to floodwaters by laterally transferring beneficial genes. To complement the 16S rRNA profiling, we have thus started viral and microbial metagenomic analyses which will identify which genes are important to degrade pesticides and recycle nutrients in the Great Barrier Reef lagoon.

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299A Halotolerant proteases from a marine bacterium: mechanism of halotolerance and tools for evaluating marine microbial ecology Kenji Aoki*1, Shinji Takenaka2 1Sagami Women's University, Japan, 2Kobe Univerisity, Japan

Halophilic bacteria produce proteases that are stable and particularly active under extremely high salt conditions. A number of halophilic proteases have been purified and characterized, and their genes have also been analyzed in view of their substantial potential in biotechnological applications. On the other hand, moderately halophilic bacteria possess halotolerant proteases that retain their activity not only under normal but also relatively high salt conditions. Since the salt concentration of seawater is around 3.5% (wt/wt) and varied according to different oceans and waters, halotolerant proteases of marine bacteria may be more favorable than halophilic proteases if they would be available for marine environment researches. Some halotolerant proteases have been purified and characterized, in order to apply them to the fermentation of certain food, but the genetic structure of the proteases have been sufficiently investigated. Thus, this study was aimed to obtain information on the halotolerance of proteases from halophilic bacteria, which will be used for evaluating marine microbial ecology.

A moderately halophilic bacterium was screened from a sea fish and identified as Bacillus subtilis. B. subtilis strain FP-133 produced two extracellular proteases, Expro-I and Expro-II, showing activity and stability at concentrations of 0-10% (wt/vol) NaCl. The two proteases were purified separately to homogeneity and characterized. The purified Expro-I was an alkaline serine protease (Apr) with an optimum pH of 7.5, although most reported serine proteases from Bacillus strains act at the higher pH than Expro-I. The molecular mass of Expro-I was 29 kDa. The purified Expro-II was a neutral protease (Npr) with the molecular mass of 34 kDa.

The N-terminal amino acid sequences of Expro-I and II showed high similarity to those of previously reported Aprs and Nprs, respectively. On the basis of conserved regions of Aprs and Nprs, PCR primers were designed and the fragments were amplified. The 401-bp and 520-bp amplified fragments corresponding to Expro-I and II genes, respectively, were used as probes for Southern analysis. The two genes were cloned. The analysis of deduced amino acid sequences showed that the mature proteins (Expro-I and II) consisted of 275 and 300 amino acid residues, respectively. The alignment analysis revealed that Expro-I had 10 acidic or polar amino acid residues conserved at sites where non-halotolerant Aprs have non-polar residues. Expro-II also had nine conserved acidic or polar amino acid residues at sites where non-halotolerant Nprs have non-polar residues. Three-dimensional modeling suggested that acidic and polar amino acid residues located on the surface stabilize protein structure in the presence of relatively high salt concentrations.

Since it was shown that the halotolerance of Expro-I and II depends on acidic and/or polar amino acid residues on the surface, we are planning to elucidate the extent or level of halotolerance by point mutation of such residues in Expro-I and II. The constructed mutants of the Expro-I and II genes expressing halotolerant proteins at various levels will be useful as probes for analyzing microorganisms in seawater, in which salt concentration varies.

300A Implementation of next-generation sequencing using Nextera on the marine cyanobacteria Prochlorococcus and Synechococcus Cecilia Batmalle*1, Sam Hsin-I2, Michael Lomas3, Adam Martiny1 1University of California Irvine, United States, 2University of California San Diego, United States, 3Bermuda Institute of Ocean Sciences, Bermuda

Prochlorococcus and Synechococcus are the most abundant photosynthetic organisms in oligotrophic waters and therefore are responsible for a significant portion of the earth's primary production. Due to their high abundance and wide distribution, many physiologically and genetically distinct lineages of Prochlorococcus and Synechococcus are commonly found in the ocean. In order to provide a view of the genetic composition of Prochlorococcus and Synechococcus population in the field, the main approach has been metagenomics. However, one of the limitations of metagenomic studies involving those cyanobacteria is the amount of input DNA needed. Here we describe the use of a novel method for High-Throughput Illumina library preparation called Nextera for metagenomic sequencing of sorted Prochlorococcus and Synechococcus populations using very low input amounts of DNA. We also present a new method for cell lysis to maximize the amount of starting DNA for the downstream

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library. The new cell lysis method involves bead beating the filter containing the sorted cells and concentrating the cell lysate by using Amicon DNA column to maximize DNA recovery. We then evaluate the lysis method using multiple displacement amplification (MDA) as a proxy to determine the amount of DNA being amplified. We found that the amount of input DNA plays an important role in determining the amplification efficiency and the quality of amplicons for downstream sequencing. We then compared the coverage and percent of sequences that matched our reference genome between Synechococcus culture samples using multiple displacement amplification followed by conventional Illumina library preparation vs. nextera library preparation without multiple displacement amplification. By looking at coverage and %sequence match, we determined that the Nextera method of library preparation showed closest results to the positive control which was genomic DNA and decided it to be our best method to analyze future environmental samples. One of the main advantages of our new method is the minute amount of input DNA needed which impedes the use a large amplification of the input DNA. Common methods for DNA amplification, such as multiple displacement amplification are known to cause certain biases including overrepresentation of higher GC content areas, high primer-primer interactions and was found to introduce bias in natural communities. We also compared the phylogenetic distribution of sorted Prochlorococcus samples and the population specific Prochlorococcus metagenomic dataset was analyzed for the presence and diversity of genes involved in phosphate and nitrogen assimilation. This includes different variants of alkaline phosphatase and newly discovered nitrate assimilation genes in Prochlorococcus. As of now this new library preparation method has not been applied to environmental marine samples. This project expands our current methods for metagenomic studies of Prochlorococcus and Synechococcus in the ocean.

301A Identification, characterization and partial purification of a novel unmodified bacteriocin from an Algerian extremely halotorant Oceanobacillus sp. strain Nadia Bouktit*1, Pierre Stocker2, Michel Fons3, Said Benallaoua4 1Université A.MIRA, Targa Ouzemour, Béjaia, Algeria, 2Institut de Chimie Radicalaire UMR 7273, Equipe sonde moléculaire en biologie et stress oxydant (ICR-SMBSO) service 522, Université Aix-Marseille, Pôle de l'Etoile, Faculté des Sciences et Techniques, Marseille, France, 3ISM2 UMR 7313, Equipe BiosCiences case 342, Université Aix-Marseille, Pôle de l'Etoile, Faculté des Sciences et Techniques, Marseille, France, 4Laboratoire de Microbiologie Appliquée, Département de Microbiologie, Faculté des Sciences de la Nature et de la Vie, Université A.MIRA, Targa Ouzemour, Béjaia, Algeria

The bottom of the deep-sea is not devoid of organisms, although the deep-sea is an extreme environment with particularly high hydrostatic pressure and low temperature. Numerous microorganisms including non-extremophilic and extremophilic microbes have been isolated from deep-sea sediment collected at depths of 10 897 m. Oceanobacillus genus was first described by Lu et al. (2001); the representative bacterium was isolated from deep-sea sediment collected at a depth of 1050 m on the Iheya Ridge; nevertheless, no antimicrobial peptide has been described for this genus until now.

We describe in this work the discovery, characterization and purification of a novel antimicrobial peptide, which is an unmodified bacteriocin produced by an Algerian extremely halotolerant Oceanobacillus sp. strain. It is a hydrophobic, plasmidencoded peptide that exhibits potent antimicrobial activity toward a number of Gram-positive and Gram-negative microorganisms, including Bacillus subtilis, Pseudomonas aeruginosa and Salmonella typhimurium. Oceanobacillus sp. strain isolated from a canned fish, was Gram-positive, aerobic, and rod-shaped.

The cell free supernatant (15% NaCl) was concentrated 100-fold (concentrated preparation CP). CP was deionised on sephadex G-25 and antimicrobial activity was confirmed using a well diffusion assay. Preparations of deionised CP were incubated with α-chymotrypsin, trypsin and papain at 37°C for 1 h at 1 mg/ml, the stability to heat was examined by heating preparations to 60, 70, 80, 90, 100°C for 10 min and at 121°C for 20 min, similarly, the stability was assessed at pH 1 to 14 by incubation in buffered solutions. Purification of the peptide was achieved using a combination of ammonium sulphate precipitation, gel filtration and reverse phase high-performance liquid chromatography.

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When examined, the bacteriocin of the Oceanobacillus sp. is completely inactivated by treatment with α-chymotrypsin and trypsin, indicating that the inhibitory substance is proteinaceous, the bacteriocin was thermostable and highly stable between pH 6 and 10. The resultant active precipitate applied to gel filtration revealed that bacteriocin had a molecular mass of approximately 50 kDa. Reverse-phase high-performance liquid chromatography analysis showed a single active fraction and Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis yielded a molecular mass of about 3.5 kDa; suggesting that the antimicrobial peptide is aggregated with other proteins, making it larger.

The bacteriocin described in this work is the first reported example of a bacteriocin produced by a strain of the Oceanobacillus genus.

302A Are potentially pathogenic Vibrio species in German coastal waters and sediments influenced by climate change? Nicole Brennholt*1, Katrin Luden2, Ernst-August Heinemeyer2, Georg Reifferscheid1, Simone Böer1 1Federal Institute of Hydrology, Germany, 2Governmental Institute of Public Health of Lower Saxony, Germany

Within the framework of the research program KLIWAS (“Impacts of Climate Change on Waterways and Navigation”) the Federal Institute of Hydrology (BfG) explores how the expected consequences of climate change such as higher average temperatures, regional increases in extreme weather events as well as changes in biotic and abiotic environmental parameters may influence water hygiene, especially transport and dissemination of microbial human pathogens and their survival and growth. Pathogenic Vibrio spp. are ubiquitous bacteria in estuarine and seawater environments and the main model organisms of our study. The genus Vibrio contains at least 12 pathogenic species, which can cause primary or secondary septicaemia. In Germany severe human infections due to Vibrio vulnificus are known since 1994. The BfG and the Governmental Institute of Public Health of Lower Saxony initiated a 2,5-year-long Vibrio spp. monitoring program in order to gain a better understanding of the ecology of potentially pathogenic Vibrio spp. in the North Sea and to answer the questions: 1) Where do potentially pathogenic Vibrio spp. occur, 2) How does the distribution and abundance of these Vibrio spp. relate to temperature, salinity and sediment type, 3) Are there “hot spots” of Vibrio spp. occurrence, and 4) Will they cause a serious health hazard in future with regard to climate warming? The results of this study will be integrated in a future-oriented health risk assessment.

Water and sediment samples were taken monthly at 11 bathing sites along the German North Sea coastline and within the estuaries of the rivers Ems and Weser. The samples were tested for V. vulnificus, V. alginolyticus, V. parahaemolyticus and V. cholerae using a culturing approach (ChromeAgar) accompanied by biochemical and molecular verification. Additionally, samples were tested for grain size, total organic carbon, temperature, conductivity and salinity.

V. vulnificus could mainly be detected at the mesohalin estuarine sites, confirming results from a lab experiment with isolates that showed limited growth at higher salinities. Positive proofs were seasonally dependent both in sediment and water. Detectable concentrations were only found at water temperatures between 14°C and 24°C, but a 20°C-threshold had to be reached first. The predominant species V. alginolyticus was found at all sites in 94% of the sediment and in 79% of the water samples, followed by V. parahaemolyticus with 67% and 44%, respectively. Presence and abundancies of V. alginolyticus and V. parahaemolyticus in the water were strongly related to temperature. Sediment Vibrios were consistently more abundant than those in the water and more resistant towards lower temperatures.

Elevated abundancies at high temperatures suggest that climate change effects – rising sea water temperatures and higher frequencies and duration of heat waves - may potentially influence Vibrio spp. distribution in Northern Europe and increase the risk of Vibrio-related wound infections by contact with sea water and sediment. Thereby, estuarine sites might be more at risk for V. vulnificus-related infections than coastal beaches. Moreover, sediments seem to serve as a potential hideaway and reservoir for these organisms in winter.

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303A Elucidating the impacts of hypoxia on the active microbes that drive nitrogen fixation in estuarine sediments Shelley Brown*1, Andraya Ehrlich1, Annaliesa Jones1, Christopher Deacutis2, Bethany Jenkins1 1University of Rhode Island, United States, 2Narragansett Bay Estuary Program, United States

Estuarine sediments harbor metabolically versatile bacteria whose activities can influence the cycle of nutrients on global scales. Microbial communities that drive nitrogen (N) cycling are extremely diverse making it difficult to identify the functional groups and elucidate controls on their activity. Recently, benthic sediments from the estuary Narragansett Bay (RI, USA) were shown to exhibit a seasonal switch in N cycling with high rates of net N2 fixation. To target the microbes driving this process, we are following the expression of a functional gene for N fixation (nifH). Organisms expressing the nifH gene in Bay sediments are relatives of Pelobacter carbinolicus and Desulfovibrio vulgaris, anaerobes that can reduce iron/sulfur and sulfate compounds, respectively. Due to nutrient loading, upper areas of the estuary exhibit episodic summer hypoxia. We wanted to elucidate the impact of changing oxygen profiles spatially and temporally on these important anaerobic N fixers and determine if the seasonal low dissolved oxygen concentrations may be a potential driver in the shift we are detecting in the N cycle. Following the microbes related to sulfur reducers we have detected the highest nifH expression during hypoxia near a wastewater treatment plant; not a N-limited environment. High rates of microbial respiration and associated hypoxic conditions may disrupt the link between coupled nitrification-denitrification, as the former is an oxygen requiring process. A shrinking oxic sediment layer and inhibition of the coupled N-removal pathway could thus expand the niche for sulfur and sulfate reducers to thrive. Nitrogen fixing by these anaerobic organisms could provide unanticipated inputs of N into environments also impacted by eutrophication, as we also hypothesize that N fixation in these bacteria is potentially insensitive to combined N sources. Field measurements of gene expression may provide insight into how these microbes react to fluctuating oxygen conditions and their contribution to inputs of N into the ecosystem.

304A The relationship between bacterial abundance, activity and growth in marine environments Barbara Campbell*, David Kirchman University of Delaware, United States

Bacteria are essential for nutrient cycling in all ecosystems. However, the contributions of individual taxa to biogeochemical cycling and how this changes over time and space is not well known. Here we describe fluctuations in taxon abundance, growth rates and activity of bacterial communities in estuarine and coastal waters using high throughput sequencing techniques (16S rRNA/rRNA gene [rDNA] tag sequencing, metagenomics, metatranscriptomics). A site in Delaware coastal waters was sampled monthly over three years. Over half of the bacterial taxa actively cycled between abundant and rare, whereas about 12% always remained rare and potentially inactive. We found that variation of dominant taxa within the SAR11 clade is most likely controlled by phosphate concentrations in the coastal environment. Abundance of one of the SAR11 taxa was correlated with phosphate concentrations. Additionally, phosphate-associated genes in the entire metagenome and in SAR11- associated genes varied seasonally, suggesting that phosphate limitation controls bacterial community structure and possible activity along the Delaware coast.

Since RNA:DNA ratios vary with growth rates in cultured bacteria, we used relative rRNA:rDNA ratios as an estimate of growth rate in situ. There was a significant correlation between the relative abundance of 16S rRNA and the relative abundance of 16S rDNA for most individual taxa during the three year study. However, 16S rRNA:rDNA ratios were significantly higher in about 20% of the taxa when they were rare than when abundant, indicating that some rare bacteria have higher growth rates than abundant taxa. Furthermore, presumed growth rates differed among the two dominant SAR11 taxa, suggesting either intraspecific competition or environmental controls on activity. In contrast, there was no positive correlation between relative abundances of 16S rRNA and rDNA for the entire bacterial community in the Delaware Bay where salinity and many other biogeochemical properties vary greatly. These data suggest that abundance of bacteria does not necessarily reflect growth rate in these estuarine waters. Presumed growth rates but not abundance of some taxa, including SAR11, reflected differences in light, nutrient concentrations (including phosphate), and other environmental factors along the estuarine gradient. These results indicate that some rare bacteria contribute

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disproportionally to biogeochemical processes. In addition, our data suggests that growth rates of individual bacteria are greatly influenced by bottom up factors in steep environmental gradients.

305A Communities of Nosz-type denitrifiers in low-oxygen waters of the Colombian Pacific Maribeb Castro-González*1, Efraín Rodríguez-Rubio2, Gesche Braker3 1Universidad del Tolima, Colombia, 2Centro de Investigaciones Oceanográficas e Hidrográficas del Pacífico-CCCP, Colombia, 3Max Planck Institute for Terrestrial Microbiology, Germany

The oxygen minimum zones (OMZ) in the Eastern Pacific are considered important sites of denitrification and N2O efflux into the ocean. Although these areas are found associated with the productive eastern boundary upwelling in the tropical North and South Pacific, enhanced N2O anomalies and slight decreases in temperature and oxygen content to 300-700 m depth have been recorded in the last fifty years at the Equatorial Pacific between 5°S to 7°N by the vertical expansion of the OMZ. The Colombian Pacific is characterized by several upwelling areas which vary throughout the year due to the presence and movement of the intertropical convergence zone and the trade winds. In the coastal area, levels of chlorophyll-a up to 4.6 mg/m3 have been reported off the mouth of Baudó river and the Tumaco Bay. The Colombian Pacific Basin shows spatial and temporal variability of temperature, salinity, density and O2 caused by the ENSO effect which is recurrent and significantly affects the physical, chemical and biological conditions in the water column. These data suggest that the activity of microorganisms related with the N cycling in this zone is important. To take into account the general lack of studies about microbial communities in this area, the goal of this research was to explore the presence and composition of denitrifying communities presumably related to N2O cycling along the Colombia Current. For this, we used the nosZ-gene that encodes N2O reductase (which has been frequently used to target denitrifying bacteria capable of the reduction of N2O to N2) along with terminal restriction fragment length polymorphism (TRFLP) analysis to uncover such communities in water samples taken between 100-500 m depths off the Pacific coast.

The TRFLP analysis shows major richness of T-RFs at 300 m with O2 levels between 3-13 µM. Denitrifying community composition was different along the Colombia current between Tumaco (02°N,79°W) and Bahía Solano (06°-77°W), and between the coast and offshore off Buenaventura (04°N-79°) at 300 m depth according to the environmental conditions. Nutrients varied along the Colombian Pacific basin and in comparison with the concentration reported in suboxic waters from tropical South Pacific. The level of nitrite was lower (0.02-0.07µM) and nitrate was higher (up to 38.9 µM) except off Tumaco Bay where low levels (6-13 µM) were recorded; ammonium was similar (0.18-0.78) except off Bahía Solano (up to 4.45µM) and oxygen was similar (3-22µM). In conclusion, our results suggest that denitrifiers with the potential to reduce N2O to N2 are present and their composition changes in the suboxic waters according to O2 levels. Although this is the first study to learn about bacteria involved in the N cycling in this area, it is necessary to expand the studies about the factors that regulates their activity, its composition and significance for N2O efflux from Colombian Pacific basin.

306A Inferring the trophic state of the Baltic Sea bacterial communities Narin Celepli*1, John Larsson1, Christopher Dupont2, Shibu Yooseph2, Johannes Goll2, Mathangi Thiagarajan2, Johannes Asplund Samulesson1, Björn Brindefalk1, Birgitta Bergman1, Karolina Ininbergs1 1Stockholm University, Sweden, 2J Craig Venter Institute, USA

Anthropogenic activities in the Baltic Sea region have directly and indirectly led to eutrophication of this brackish water body, which in extension has caused anoxia and subsequent 'suffocation' of macrozoobenthos, as well as fueled phytoplankton growth. Whether or not eutrophication has increased the occurrence of phytoplankton blooms, or if phytoplankton blooms - the primary concern being toxic cyanobacterial blooms in the Baltic proper - have increased at all from a historical perspective, is an ongoing debate. It is reasonable, however, to suspect that the composition of the microbial communities of the Baltic Sea responds to prevailing nutrient concentrations. It was recently shown that diversity of Baltic Sea microbes do not follow the Remane curve, i.e. a minimum of species richness in brackish water, which has been observed for animals and mesozooplankton. It has so far not been shown if this pattern is also reflected in functional diversity.

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To assess the trophic state of the Baltic Sea bacterial communities, metagenomic data from a transect of 21 samples from 11 stations sampled during the 2009 Global Ocean Sampling (GOS) expedition was used, ten from the Baltic Sea and one from the landlocked freshwater lake Torne Träsk in northern Sweden. The occurrences of 123 strains of bacteria, classified as either copiotrophic (52) or oligotrophic (71) in published literature, were mapped at the stations in the sampled transect, and used as proxies for assessing the trophic state of the bacterial communities of the Baltic Sea and surrounding waters.

Of all 123 strains (or closest higher taxon), 94 were found in the Baltic Sea GOS samples; of these, 48 were oligotrophic and 46 were copiotrophic. Oligotrophic species were more common in more saline waters (13.7-34.4 psu) on the Swedish West coast, whereas copiotrophic species were more common in less saline waters (0-4.5 psu), mainly from the Gulf of Bothnia and Torne Träsk. However, a subset of six samples, mainly from the brackish Baltic proper, showed no dominance of either trophic category, suggesting that the bacterial communities in this eutrophic basin are adapted to both variable and rapidly changing (typical for copiotrophs) and more static (typical for oligotrophs) conditions. This suggests that the unexpected diversity of brackish water microbes is reflected also in a functional diversity, with an unusual adaptation to both copio- and oligotrophic lifestyles.

307A Cold endured, low light adapted and diverse picocyanobacteria isolated from the winter season of Chesapeake estuary Feng Chen*1, Yongle Xu2, Nianzhi Jiao2 1University of Maryland Center for Environmental Science, United States, 2Xiamen University, China

Our recent studies in the Chesapeake Bay showed that picocyanobacterial community structure and abundance change dramatically from summer to winter. Many picocyanobacteria from warm seasons of Chesapeake Bay have been isolated and studied, but none has been isolated from the winter time. This study is set to isolate picocyanobacteria from the cold winter of Chesapeake Bay and understand the physiology of cold-adapted picocyanobacteria living in a temperate estuary. Fifteen strains of picocyanobacteria were isolated/obtained from Baltimore Inner Harbor from December 2010 to February 2011. The water temperature ranged from 2-8 °C and salinity from 4.5-22.0‰ during this period. The winter isolates belong to five phylogenetic lineages based on the 16S rRNA gene and ITS phylogeny. Eight strains clustered with the Bornholm Sea cluster, and 4 strains with the Subalpine cluster. The 3 remaining strains do not have closely related culture counterparts and appear to be novel picocyanobacteria. Many winter strains grew slowly at 4 °C, but the summer strains showed negative growth at low temperature. The winter isolates grew in a wide range of salinity (0 to 40 ppt), showing a stronger salt tolerance capability compared to marine strains like WH7803 and WH7805. Cultures with yellow brown or dark red color were commonly seen among the winter collection. The winter isolates prefer to grow under dim light (3 µE/m2/s), and the light intensity of 20 to 30 µE/m2/s appeared to inhibit their growth on the microplates. None of picocyanobacteria isolated in winter were closely related to those isolated in summer. We demonstrate that despite present in low abundance diverse picocyanobacterial populations are present in cold winter. The winter picocyanobacteria grow slowly at low temperature and are more adapted to low light intensity. It would be interesting to learn if these winter isolates share similar ecophysiological features with polar picocyanobacteria.

308A Mixing of water masses caused by a drifting iceberg affects substrate utilization and community composition of bacterioplankton in the Southern Ocean Julie Dinasquet*1, Inga Richert2, Ramiro Logares3, Stefan Bertilsson4, Lasse Riemann5 1Linnaeus University - Copenhagen University, Denmark, 2Uppsala University - Helmholtz Centre for Environmental Research, Sweden, 3Institure of Marine Sciences, CSIC, Spain, 4Uppsala University, Sweden, 5Copenhagen University, Denmark

Increased formation of drifting icebergs through ice-shelves disintegration has been observed over the past decade in Antarctica. Water column mixing caused by drifting icebergs influences local physical and chemical parameters, affects food web composition and leads to increased productivity and possibly increased burial via the biological carbon pump. Bacterioplankton are known as main determinants of the fate of organic carbon in the sea; however, data on whether icebergs affect local bacterioplankton function and composition are scarce.

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In this study we assessed the influence of an iceberg on local bacterial community composition and the ability of bacterial assemblages to exploit a range of carbon substrates. During the Antarctic summer 2010/11 in the Amundsen Sea Polynya, we sampled an area influenced by a drifting iceberg as well as a control area with undisturbed water column. Sterile-filtered seawater was obtained from the Deep Chlorophyll Maximum (10 to 40 m) and from deeper waters (300 to 400 m) at each area, inoculated with a 20% v/v bacterial inoculum from the respective depths, and amended with inorganic nutrients and carbon substrates (a mix of substrates, glutamine, N-acetylglucosamine (NAG), or pyruvate). Triplicate 1-L bottles for each treatment were incubated for a week in darkness and at in-situ temperature. Bacterial abundance and production were followed over the course of the experiment and bacterial community composition at the start and end of the experiment was examined by 454-pyrosequencing of 16S rRNA gene.

An elevated bacterial production and a distinct community composition were observed in the iceberg influenced waters compared to the control. Surprisingly, a strong growth response to specific substrates as well as a distinct community succession were observed only in the area where the water masses were influenced by iceberg mixing. For example, relative to control incubations, the addition of NAG to deep water from the iceberg-influenced area stimulated the growth of Polaribacter (Bacteroidetes) at the expense of γ-proteobacteria-related groups while the addition of pyruvate stimulated the growth of Colwelliaceae (γ-proteobacteria) at the expense of Oceanospirillales (γ-proteobacteria) and Polaribacter.

In summary, this study shows that drifting icebergs can affect bacterioplankton community composition as well as community function; i.e. the capability to utilize specific carbon substrates. Hence, in a climate change scenario, an increased frequency of drifting icebergs in the Southern Ocean may affect the efficiency of the biological pump.

309A Evidence for complex lifestyles among aquatic bacteria: Switching microhabitats reversibly changes metabolic capacity of aggregate-associated microbial communities Fred C. Dobbs*1, M.M. Lyons1, X. Zhao2, R.E. Hicks2 1Old Dominion University, Norfolk, Virginia, USA, 2University of Minnesota-Duluth, Duluth, Minnesota, USA

Suspended particles, including organic aggregates, represent discrete microhabitats within the water column and, as a result, partition microbial communities into consortia dominated by particle specialists and free-living specialists. Using Biolog Ecoplates to measure metabolic capacity (e.g. metabolic activity, functional diversity, and percent nitrogen use), we provide support for the importance of an intersecting category (i.e. particle generalists) by demonstrating short-term (<24h) reversible changes in community metabolic capacity as a function of microhabitat. Overall, metrics of metabolic capacity were 2 to 3 times higher and 5 to 9 times less variable for attached communities compared to free-living ones, supporting the concept that organic aggregates provide more favorable and more consistent habitat for associated copiotrophic microbes. We grouped heterotrophic bacteria into four functional groups based on life mode: particle specialists (PS), particle generalists in the aggregate fraction (PG-A), particle generalists in the water fraction (PG-W), and free-living specialists (FLS). We demonstrated that on average, PS and PG-A recorded significantly higher utilization of amino acids, carbohydrates, carboxylic acids, and nitrogen-containing carbon resources than did PG-W and FLS. Utilization of polymers did not differ significantly among life modes due to highly variable utilization of lipid-like polymers by PS and PG-A compared to utilization of sugar-like polymers by PG-W and FLS. Metabolic capacity at this microscale is important in the context of biogeochemical cycles, microbial-loop food web dynamics, and virulence of aquatic pathogens, and supports the role of organic aggregates as island-like microhabitats for heterotrophic bacteria.

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310A Diversity and seasonal dynamics of haptophytes in outer Oslo fjorden as revealed by 454 pyrosequencing Elianne Egge*1, Wenche Eikrem1, Tom Andersen1, Vladyslave Hostyeva1, Lucie Bittner2, Torill Johannesen3, Ruth-Anne Sandaa3, Bente Edvardsen1 1University of Oslo, Department of biology, Norway, 2University of Kaiserslautern, Department of ecology, Germany, 3University of Bergen, Department of biology, Norway

Haptophytes constitute an important part of the marine plankton community and play a major role as primary producers. Bloom formers may have large biogeochemical and economic impact through production of toxins harmful to marine biota and carbon sequestration through calcite scale formation in coccolithophores. Knowledge on the biodiversity and seasonal dynamics of haptophytes at the species level is limited because species identification often requires electron microscopy or molecular methods. Here we present results from a study using 454-pyrosequencing to investigate the seasonal dynamics of the haptophyte community in outer Oslofjorden. Samples of the subsurface nano-and picoplankton were collected monthly for two years, and the haptophytes were targeted by amplification with group-specific SSU rDNA V4 primers. The pyrosequencing data were supplemented with clone libraries and microscopy. Considerable diversity is revealed, particulary within the order Prymnesiales. Members of more than 20 genera were detected, distributed among all 8 known haptophyte orders, The pyrosequencing data retrieved species that have not previously been observed by microscopy in the area, and also clades consisting only of hitherto uncultured species (clade D and E). Analysis of the diversity in relation to physiochemical factors is in progress to shed light on mechanisms driving changes in haptophyte community structure.

311A Microbial degradation of organic matter under elevated pCO2: an offshore mesocosm study Sonja Endres*, Luisa Galgani, Anja Engel Helmholtz Centre for Ocean Research Kiel (GEOMAR), Germany

Extracellular enzymes process organic matter degradation as well as nutrient regeneration and hence play an important role in the turnover of dissolved organic matter (DOM). Ocean acidification is expected to affect enzymatic hydrolysis, resulting in changes in microbial decomposition of organic matter.The effects of increasing CO2 concentrations on the natural planktonic community, bacterial exopolymer degradation, and particle export were studied during a joint SOPRAN/BIOACID mesocosm experiment in the Raunefjord in southern Norway. Here, we report on the effect of pCO2 on production of transparent exopolymer particles (TEP) and exo-enzymatic degradation.

Nine 25m-long Kiel Off-Shore Mesocosms for Future Ocean Simulations (KOSMOS) were adjusted to different pCO2 levels ranging from ca. 280 to 3000 µatm by stepwise addition of CO2 saturated seawater. After CO2 addition, samples were taken every second day for 34 days. The first phytoplankton bloom developed around day 5. On day 14, inorganic nutrients (5 µmol L-1 nitrate and 0.1 µmol L-1 phosphate) were added to the enclosed, nutrient-poor waters to stimulate a second phytoplankton bloom, which occurred around day 20. We determined bacterial cell counts, rates of extracellular enzyme activities using fluorescent-labelled substrate analogues, as well as concentration and composition of organic matter. We observed increasing bacterial cell numbers and TEP concentrations over time with highest increase in the high pCO2 treatments. Preliminary results show higher extracellular enzyme activities at higher CO2 concentrations. Positive effects of ocean acidification on the bacterial community and especially on extracellular enzymes may potentially increase bacterial degradation activity and consequently the release of CO2 to the atmosphere in the future ocean.

312A The mystery of the unknown: patterns in the global deep ocean heterotrophic bacterioplankton activity Ana Gomes*1, Xosé Anxelu G. Morán2, Josep M. Gasol1 1Institut de Ciències del Mar (ICM), CMIMA - CSIC, Spain, 2Centro Oceanográfico de Gijón, Instituto Español de Oceanografía, Spain

The deep ocean constitutes the largest habitat in the biosphere yet it is still unexplored in its majority. Based on low temperatures and substrate availability, a postulated large decrease in all biological

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processes with depth pictures a deep oceanic realm with almost negligible activity. Recently, this assumption of a "dead" deep-sea has become questioned by increasing data that show deep-ocean Bacteria and Archaea to be growing at rather high rates. Open ocean samples were collected during the worldaround Malaspina Expedition in 2010-2011 from the sunlit epipelagic waters to the dark bathypelagic zone, covering the major oceanic basins of the global ocean including the North and South Atlantic, the Pacific and the Indian Ocean. Preliminary results indicate higher total abundances of heterotrophic prokaryotes (Bacteria and Archaea) in deep North Atlantic waters (4×105 cells mL-1) as compared to the other basins (8×103 cells mL-1 in the South Atlantic and 3×104 cells mL-1 in the Indian and Pacific Oceans). Despite these basin-scale differences, a common >1 order of magnitude decrease in abundance from the photic zone to the dark ocean was observed. We also measured single cell and bulk activities which point to significant activity levels in deep waters of all the major basins. Using archaeal inhibitors of bacterial activity, we show that Archaea contribute to approximately 40% of total heterotrophic prokaryotic production in the deep oceanic waters.

313A Occurrence of antibiotic resistance in marine sediments and fauna along an exposure gradient in Arctic Greenland Maria Granberg*, Ingela Dahllöf University of Gothenburg/Dept. of Biology and Environmental Science, Sweden

The presence of antibiotic/antimicrobial substances in the marine environment is of high concern since they potentially accelerate widespread bacterial antibiotic resistance (AR), and may affect natural bacteria communities responsible for sustaining vital ecosystem functions. AR bacteria associated with marine organisms can potentially travel along marine food chains and reach commercial species, for example shellfish, fish and mammals. Mapping AR in different habitats is therefore urgent to fully understand the extent and roles of environmental resistance reservoirs. In Greenland, sewage treatment is generally lacking and municipal wastewater is discharged directly into the ocean, potentially creating strong gradients of pharmaceuticals along the town coasts.

During the summers of 2009 and 2010, we placed passive samplers for antibiotics at five sites around the town of Sisimiut, Greenland. Two sites were located at major sewage outlets, and the other three at varying distance from the outlets. Mussels, fish and sediment were collected at the different sites. Bacteria were cultured from gut tissues and sediment samples, and tested for resistance towards four different antibiotics, Benzylpenicillin, Amoxicillin, Roxithromycin and Ciprofloxacin. Bacterial colonies were identified using API-tests targeting human enterobacteria.

There was a strong gradient of resistance from point sources to more open waters for all antibiotics, but the magnitudes of resistance and the resistance fingerprints differed among sites. Even at the most distant site, resistance towards the more synthetic antibiotics was present. There was also a parallel gradient in the controls with respect to the number of colony forming units, indicating a lower bacterial gut content in fish and mussels closer to sewage outlets. Resistant bacteria from samples collected closer to the sewage outlet were more often identified as human enterobacteria than resistant bacteria obtained further from the sources.

Results describe a strong impact of human activities on bacterial AR development in the marine environment around Greenland. Since Greenlandic people traditionally depend heavily on sea-food as a food source, the presence of AR bacteria found in guts of fish and shellfish poses a threat to the people in terms of acquiring AR-genes and AR. To our knowledge this is one of the first field investigations of antibiotics and resistence development in the marine Arctic environment.

314A Bromodeoxyuridine incorporation techniques reveal actively growing bacteria in a temperate coastal water of Hiroshima Bay, western Japan Koji Hamasaki*1, Yuya Tada1, Akito Taniguchi2 1Atmosphere and Ocean Research Institute, Japan, 2Kinki University, Japan

Bacterial productivity and community structures have been intensively studied to understand ecosystem structures and biogeochemical cycles in the ocean. Yet, the fundamental questions that persist are "which species account for bacterial productivity?" and "what is the relative contribution of each?" Quantitative evaluation rather than qualitative description of bacterial community functions are important to model food web dynamics and biogeochemical cycles. We have been developed

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bromodeoxyuridine (BrdU) incorporation methodologies to label actively growing bacteria and relate bacterial productivity to their community structures. There are three BrdU methodologies. Firstly, total bacterial production is measured by quantifying bulk incorporation of BrdU with the use of membrane dot blotting and chemiluminescense antibody detection. Secondly, bacterial genomic DNA is extracted from filtered samples and then BrdU-labeled fraction is immunologically separated from total DNA. BrdU-labeled DNA is subjected to DGGE fingerprinting analyses after amplifying 16S rRNA genes. Sequencing excised DGGE bends shows phylogenetic identities of BrdU-incorporating (presumably actively growing) bacteria. Thirdly, based on the 16S rRNA gene sequences of actively growing bacteria, DNA probes are designed for fluorescence in situ hybridization (FISH). BrdU immunocytochemistry in combination with FISH enables the count of specific groups of actively growing bacteria and the estimation of their relative contribution to total bacterial production. In our pilot study, three methods were successfully integrated to monitor temporal variabilities of bacterial productivity and community structures. A year-round monthly sampling was performed in a temperate coastal water of Hiroshima Bay, western Japan. We identified 23 phylotypes of actively growing bacteria, belonging to Alphaproteobacteria (Roseobacter group, 9 phylotypes), Gammaproteobacteria (2 phylotypes), Bacteroidetes (8 phylotypes), and Actinobacteria (4 phylotypes). The Roseobacter group and Bacteroidetes were dominant and together accounted for more than 70% of the total DGGE bands. We revealed that community structures of actively growing bacteria shifted markedly at the timing of phytoplankton blooms. The year-round monitoring through BrdU immunocytochemistry-FISH revealed the importance of Roseobacter/Rhodobacter group, which was found to be the constantly proliferating population (27% of BrdU-positive cells). Their abundance however, was not significantly correlated with water temperature or with chlorophyll a or, organic matter concentrations. Bacteroidetes group was another important group, as they greatly increased in abundance after the end of phytoplankton blooms. Two other phylotypes tested in this study, the SAR86 and Vibrio groups, changed their activities/abundance correspondingly with water temperature. Our study revealed the usefulness of the BrdU methodologies to monitor bacterial growth and productivity in seawater environments. Future studies should be focused on the specific functions possessed by these actively growing bacteria. For example, metagenomic analysis in combination with the BrdU incorporation technique can be very promising to reveal potential functions of actively growing bacteria in addition to their phylogenetic affiliations. Such study is fundamental to understand biogeochemical processes in marine ecosystems.

315A Temperature dependence of prokaryotic autotrophy in the meso- and bathypelagic Atlantic Ocean Roberta Hansman*, Gerhard Herndl University of Vienna, Austria

Chemoautotrophy in the meso- and bathypelagic ocean has recently been identified as a significant metabolic pathway of prokaryotes comparable to the magnitude of heterotrophic prokaryotic metabolism. While some studies have implicated the oxidation of ammonia by members of Thaumarchaeota as fueling dissolved inorganic carbon (DIC) fixation, the extent and potential of in situ energy sources for chemoautotrophy in the dark ocean have not been fully identified and quantified. Seawater collected in the meso- and bathypelagic eastern Atlantic Ocean and incubated at 20˚C exhibited DIC fixation rates up to 500 times greater than rates measured at in situ temperatures. These high rates of autotrophy are not accompanied by an increase in prokaryotic abundance, indicating a probable gene expression response by organisms present in situ. As these incubations were amended solely with 14C-labeled bicarbonate, it appears there are potential energy sources available in the dark ocean supporting high levels of chemoautotrophy that are not, or not efficiently, utilized at ambient temperatures. In incubation experiments, the temperature dependence of the DIC fixation as well as the accompanying changes in microbial community composition were determined in combination with a metagenomic approach to further constrain the chemoautotrophic potential of the deep ocean.

316A Comparative community genomics of the Northeast subarctic Pacific Ocean Niels Hanson*, Jody J. Wright, Kishori M. Konwar, Steven J. Hallam University of British Columbia, Canada

Environmental genomics captures the diversity and dynamics of naturally occurring microbial communities. The metabolic potential of an organism or community can be inferred from primary sequence information with the aid of computational methods that assemble or cluster contiguous

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reads, search for patterns or motifs representing genes, and reconstruct metabolic pathways based on known biochemical rules. As individual microbial genomes are mosaics of genes of mixed heritage, a microbial community is a collection or network of gene functions distributed amongst its individual members. Understanding how these networks form, function and change over time reveals otherwise hidden linkages between microbial community structure and higher order ecological and biogeochemical processes. Although next generation sequencing technologies are rapidly expanding our capacity to uncover these networks, a number of outstanding computational challenges limit full data utilization and interpretation. Here we apply a novel analytic pipeline (MetaPaths) that adopts Pathway Tools (http://bioinformatics.ai.sri.com/ptools/ptools-overview.html) for reconstruction of microbial community structure and function in the oxygen minimum zones (OMZ) of the Northeast subarctic Pacific Ocean (NESAP). Pathway Tools is a reusable, production-quality software environment for creating and managing pathway/genome databases (PGDBs) and predicting interaction networks based on a highly curated database of metabolic pathways and components representing all domains of life (MetaCyc). Comparison of 35 PGDBs representing different depths and times of year along a coastal to open ocean transect of the NESAP revealed distinctive patterns of redox-driven niche partitioning between oxic surface and core OMZ waters. Pathways for sulfur, ammonia, and methane oxidation as well as sulfate reduction and inorganic carbon fixation were prevalent and co-varied between upper and lower oxycline and core OMZ. Despite depth interval co-variation, spring, summer and winter depth profiles were ~72% similar in pathway composition consistent with a stable functional core within NESAP waters. Moreover, unique pathways were more often associated with surface waters consistent with bloom dynamics. These results provide an ecological blueprint for understanding the metabolic interactions mediating matter and energy transformations in the largest permanent OMZ in the global ocean and a baseline for monitoring the impact of climate change on microbial community metabolism.

317A Characterization of microbial communities through space and time after oil spill in Bohai bay Xiaoke Hu*, Hui Wang, Jialong Yang Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, China

Increasing exploitation, production, transportation, and storage of oil at sea have led to more accidentally spilled contaminants. The oil spill in the Bohai bay is the deepest and largest offshore spill in China and its impacts on marine ecosystems are still largely unknown. The microorganisms are known to have predominant roles in degradation of oil contaminants. The hypothesis is that the microbial community functional composition and structure will be dramatically altered after the oil spill. The investigation of the microbial community diversities through space (locations) and time (seasons) will increase the understanding of bioremediation functions. Therefore, the metagenomic techniques were applied to investigate the complex microbial communities in Bohai oil spill area. The seawater and sediments samples from various contaminated sites in two different seasons were collected and analyzed for the distribution of contamination by HPLC and GC-MS. Denaturing gradient gel electrophoresis (DGGE) and clone library techniques were used to analyze the changes of the functional microbial communities. While, quantitative real-time PCR (qRT-PCR) were conducted to analyze the abundance of the microbial communities. For the metaproteomic study, the total intracellular and extracellular proteins were extracted and quantified from seawater and sea sediments and then separated by 2D-PAGEs. The image master system was used to analyze the differential proteins. MALDI-TOF-MS were conducted to obtain peptide mass fingerprints. The differential proteins selected and excised from 2D-PAGE gels were identified by comparing the SWISS-PROT using Mascot algorithm. The results indicated that the marine microbial communities changed through space and time and could have a significant role in biodegradation of oil spills after the massive oil spill occurred.

318A Characterization of sulfate-reducing communities using dissimilatory sulfite reductase (dsr) gene in the finfish (Rock fish and Seabream) aquaculture farm in the southern coast of Korea Jung-Ho Hyun*1, Ayeon Choi1, Hye-Yeon Cho1, Bomina Kim1, Sunguk An1, Won-Chan Lee2 1Hanyang University, South Korea, 2National Fisheries R&D Institute, South Korea

Global production of food fish from aquaculture, including finfish, crustaceans, and mollusks, had been increasing from 2.6 million tonnes in 1970 to 52.5 million tonnes in 2008, which accounted for 45.7 percent of the world's fish food production for human consumption in 2008. Similarly, in Korea,

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aquaculture production had been doubled from 65 million tonnes in 2000 to 139 million tonnes in 2007. Especially, the finfish production had been tripled from 26,000 tonnes in 2000 to 98,000 tonnes in 2007. However, excess deposition of organic enriched fish food and feces on the sediment surface may cause serious environmental impact by enhancing anoxic conditions. In anoxic marine sediment, microbial sulfate reduction is regarded as a major carbon oxidation pathway, thereby accumulating toxic HS- in the sediment and releasing inorganic nutrients into overlying waters. Here, we report biogeochemical conditions, sulfate reduction rates (SRR) and abundance and diversity of dissimilatory sulfite reductase (dsr) gene associated with sulfate reduction together with the abundance of prokaryotes using 16S rRNA gene in the caged finfish (Rock fish and Sea bream) farm of the southern coast of Korea.

Geochemical parameters in the farm site exhibited highly reduced condition; HS- and NH4+ in the pore

water of the farm sediment (11.8 mmol S m-2 and 106 mmol N m-2) was 16 times and 1.5 times, respectively, higher than that of the reference site (0.62 mmol S m-2 and 72 mmol N m-2). Accordingly, SRR was 20 times higher at the farm site (123 mmol S m-2d-1) than that at the reference site (6.2 mmolS m-2d-1). Depth-integrated prokaryotic cell number estimated from 16S rRNA gene copy number was similar at both farm (13.6 x 1014 cells m-2) and reference (9.9x1014 cells m-2) sites, but the abundance of sulfate reducing prokaryotes (SRP) estimated from dsr gene copy number was 3 times higher at the farm site (1.2x1014 cells m-2) compared to the reference site (4.0x1013 cells m-2). Composition of SRP communities revealed from the dsr gene libraries appeared similar at both farm and reference sites. The most predominant members of SRP were affiliated to Syntrophaceae group (61% and 65% of total dsr gene at the farm and reference sites, respectively) that is known to degrade propionate using sulfate as an electron acceptor. The second dominant SRP group comprising 25% of total dsr gene clones at both sites was not affiliated to known SRP. This novel group was closest to the clone TopDsr59 detected in Pearl River sediment (similarity, 87%). Desulfobacter-Desulfobulbus was detected as third dominant group (14% and 10% at the farm and reference sites, respectively). Overall, fish farm sediment was characterized by remarkably reduced geochemical condition and enhanced abundance of SRP and SRR. Pre-dominance of incomplete organic matter oxidizing SRP (i.e., Syntrophaceae and Desulfobulbus) further indicated that microbial composition of SRP at both sites were highly affected by excess loading of artificial organic matter such as fish food.

319A Geographical differentiation of the genome content of the marine cyanobacterium prochlorococcus Alyssa Kent*, Adam Martiny University of California, Irvine, USA

Prochlorococcus, the smallest known photosynthetic bacterium, is ubiquitous in the ocean’s surface layer despite variation in ecological conditions. Within the species, there are several genetically distinguishable ecotypes allowing it to persist in diverse environments. Vertical or horizontal transfer of genetic information drives changes in genomic content, however it is still unclear how significant each of these mechanisms is for the distribution of Prochlorococcus diversity. Is the genomic diversity of Prochlorococcus population equally influenced by vertical or horizontal evolution, or is one mechanism favored over the other? Does the preference depend on the function of the gene and its relation to the environmental factors? In order to begin to address these questions, we identified genes from molecular data acquired from the Global Ocean Sampling Expedition. We first identified sequences highly similar to known Prochlorococcus genes and used geographic data to define the spatial distribution of orthologous genes. On average, 618 non-core genes (those not found in all of the 13 fully sequenced Prochlorococcus genomes) are present in each region, but the composition of genes differs geographically. Samples were hierarchically clustered based on Bray-Curtis similarity of orthologous gene abundance. As expected, samples were more similar in the set of core genes than the set of non-core genes. Furthermore, samples clustered predominantly based on geography. Analogous to gene expression profiling studies that identify groups of genes expressed similarly under specific conditions, we profiled orthologous gene group abundances based on different ecological parameters, such as geography; temperature; and levels of phosphate, nitrate, and iron in the sample. We examined a case study of one functional group of known genes related to phosphate uptake. They predominantly clustered together and defined a group of genes with similar patterns across different samples. Discovering the relationship between groups of orthologous genes and the abiotic factors controlling their expression gives us a better understanding of the components controlling the gene abundance of sampled Prochlorococcus.

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320A The Baltic Sea microbiome exposed by large-scale environmental sequencing John Larsson*1, Karolina Ininbergs1, Johan Nylander1, Narin Celepli1, Jarone Pinhassi2, Chris Dupont3, Shibu Yooseph3, Johannes Goll3, Mathangi Thiagarajan3, Birgitta Bergman1 1Stockholm University, Sweden, 2University of Kalmar, Sweden, 3J. Craig Venter Institute, USA

The Baltic Sea is a semi-enclosed water body with several large sub-basins separated by shallow waters. Salinity levels range from low (2-3 PSU) in the north Bothnian Bay to intermediate (6-8 PSU) in the Baltic Proper, making this body of water one of the largest brackish systems in the world. In addition, the sea is connected to fully limnic environments via major riverine input from large rivers (in the north) as well as to marine waters (the North Sea) via belts around Denmark in the south-west. Being in the temperate climate zone this aquatic ecosystem is also subject to drastic seasonal variations (for example temperature, light and nutrient abundance), with long periods of ice coverage stretching from the Bothnian Bay in the north towards the Baltic Proper, alternated by shorter periods of warm water temperatures during the summer.

With a catchment area of approximately 90 million people, the Baltic Sea is more influenced by anthropogenic activities than most marine environments. Sources of pollution include industry and municipal wastes as well as deposits from maritime activities. Additional pollutants include mercury, PCBs and dioxins. Eutrophication, resulting primarily from increased loads of nitrogen and phosphorous from modern agricultural practices, has also increased the environmental stress on the Baltic Sea and caused oxygen depletion in bottom sediments, severely impacting the life in the Baltic Sea. Given this increased pressure on the Baltic Sea biota we sought to obtain a holistic view of the microorganisms which make up the food-web of this ecosystem. Through metagenomic sequencing of samples collected at 11 geographic locations along a 2,500 km long transect (part of the European leg of The Sorcerer II Global Ocean Sampling expedition in July 2009), including marine (eastern North Sea), brackish (Baltic Proper, Bothnian Sea and Bothnian Bay) and limnic environments (lake Torne Träsk in the north of Sweden) we are able to map the composition and functional properties of the Baltic Sea microbiome in an unprecedented manner. Serial filtration and depth profiling resulted in 70 metagenomic libraries of approximately 24 million peptide sequences, including viral fractions, which we relate to other marine and fresh-water ecosystems.

We present a microbiome highly structured by environmental gradients which includes novel members with adaptive capabilities required by this dynamic ecosystem.

321A High frequency sampling of bacterioplankton communities reveals seasonal dynamics in the baltic sea – implications for long-term shifts in climate Markus V. Lindh*1, Anders F. Andersson2, Emmelie Nilsson1, Federico Baltar1, Catherine Legrand1, Jarone Pinhassi1 1Marine Microbiology, School of Natural Sciences, Linnaeus University, Sweden, 2KTH Royal Institute of Technology, Science for Life Laboratory, School of Biotechnology, Sweden

Studies of marine bacterial dynamics have revealed the potential of bacterial communities to respond to natural or anthropogenic disturbances on both spatial and temporal scales. So far there are few, if any, predictable patterns of responses in bacterial diversity and ecosystem function to environmental disturbance. There is a need to better understand the connection between the development of specific bacterial lineages (at different phylogenetic levels), physiological capacity and environmental fluctuations. Seasonal dynamics of bacterioplankton communities were investigated by high-frequency sampling (twice weekly) from March to December 2011 in the Baltic proper. Temperature, salinity, nutrients and chlorophyll a were measured, together with bacterial abundance (by flow cytometry) and bacterial heterotrophic production (via leucine incorporation). Phytoplankton biomass (chlorophyll a), showed a typical seasonal pattern for this region, with peaks in April (that is diatom and dinoflagellate bloom) and July (that is cyanobacteria bloom). Furthermore, these peaks in phytoplankton biomass were closely associated with increases in bacterial heterotrophic production. Pronounced temporal dynamics were also observed in bacterial abundance (specific peaks were observed in April, June and August) and populations detected by flow cytometry. Collection of DNA and subsequent analysis by deep 454 pyrosequencing of amplified 16S rRNA gene fragments revealed significant compositional changes during the study. Temperature, water circulation patterns and algal blooms were major

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drivers of shifts in bacterial community composition, where specific bacterial populations (operational taxonomic units) were coupled with peaks in bacterial production and abundance. For example, at times of cyanobacterial blooms, specific populations of Bacteriodetes and Gammaproteobacteria became abundant, at the expense of a diversity of Alphaproteobacteria populations. Moreover, Betaproteobacteria dramatically increased when temperature decreased again in winter at around 0.5-4°C. Our results indicate that temporal dynamics of marine bacteria in the Baltic Sea can be classified into: (i) resilient populations present throughout the year, continuously in high abundance but variable in response to environmental fluctuations, (ii) neutral populations present throughout the year, but continuously in low abundance and non-responsive to environmental fluctuations, and finally (iii) opportunistic intermittent populations originating from the rare biosphere that appeared and disappeared depending on environmental fluctuations. Surprisingly, this response of bacterioplankton populations to environmental fluctuations were resolved at different phylogenetic scales, where for example, the temperature response was resolved at a broad phylogenetic scale (95% 16SrDNA similarity). In conclusion, our study provides a framework for understanding the relationship between bacterioplankton phylogeny at different scales, physiological capacity and ecosystem response to natural and human induced environmental changes.

322A Abundance, expression, and diversity of nitrite reductase genes in Gulf of California and eastern tropical North Pacific oxygen minimum zones Marie Lund*, Jason M Smith, Jassica A Lee, Christopher A Francis Stanford University, United States

In oxygen minimum zones (OMZs), ammonia oxidation provides a source of oxidized N that can be used as an electron acceptor by anammox bacteria and denitrifiers under anoxic conditions and converted to gaseous N forms in the process. Nitrite is a key intermediate in this nitrogen cycle, providing a critical link between the aerobic ammonia oxidation process and the anaerobic processes of denitrification, anammox, and dissimilatory nitrate reduction to ammonia (DNRA). We investigated the abundance and diversity of nitrite reductase genes linked to all of these processes in an oceanic transect across the 6 stations in the OMZ in the Gulf of California (GOC) and the eastern tropical North Pacific (ETNP).

Abundance of denitrifiers was measured via qPCR analysis of nirS (nirK was undetectable), anammox bacteria were detected through Scalindua-type nirS, and DNRA organisms were screened for (unsuccessfully) via nrfA. In addition, the abundance, expression and diversity of two nirK variants (AnirKa and AnirKb) from ammonia-oxidizing archaea (AOA) was investigated along with two ecotypes (water column A and B) of AOA ammonia monooxygenase subunit A (amoA).

All known denitrifiers are facultative aerobes and denitrifier nirS (DNF-nirS) was therefore expected to be present throughout the water column; this was indeed the case, although an increase in abundance was observed in the suboxic part of the water column. Phylogenetically DNF-nirS sequences fell in two major groups together with sequences previously retrieved from the Arabian Sea and eastern South Pacific OMZs, but biogeographic patterns were not observed.

Anammox Scalindua-type nirS (Sc-nirS) was only present in the suboxic part of the water column. The Sc-nirS sequences formed two clusters together with sequences form the Peruvian OMZ and sediment sequences from the South China Sea. A biogeographic signal was apparent in the Sc-nirS phylogeny, with sequences from the GOC and the ETNP roughly divided into each respective cluster.

The function of nitrite reductases in ammonia-oxidizers is largely unknown, but the nirK gene is consistently present in both bacterial and archaeal ammonia-oxidizers; furthermore, marine AOA carry a phylogenetically distinct type of nirK. It has been speculated that NirK is (i) indirectly involved in ammonia oxidation or (ii) perhaps nitrite detoxification. In the GOC and ETNP, the nirK gene co-occurrs with AOA amoA and both genes are present throughout the water column, with a peak in abundance just below the photic zone where the water column is still oxic. The expression of both genes (both in absolute numbers of cDNA copies and when normalized to gene copy numbers) is also highest just below the photic zone, coinciding with where the highest ammonia oxidation rates were previously measured in the same water body. On average, AOA nirK is 2.7-fold more highly expressed than amoA, suggesting that this gene is indeed of physiological importance in AOA. No patterns in nirK or amoA diversity were observed with respect to oxic versus suboxic zone or geographic location.

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323A Dissolved organic matter and sea ice dynamics may explain changes in microbial community structure along the Western Antarctic Peninsula Catherine Luria1, Linda Amaral-Zettler2, Hugh Ducklow3, Daniel Repeta4, Jeremy Rich*1 1Brown University, USA, 2Josephine Bay Paul Center, Marine Biological Laboratory, USA, 3Ecosystems Center, Marine Biological Laboratory, USA, 4Woods Hole Oceanographic Institution, USA

The Western Antarctic Peninsula (WAP) experiences extreme seasonal changes in sea-ice and light, with intense phytoplankton blooms in spring and summer. Microbial communities in this region and their coupling to phytoplankton dynamics remain poorly understood. To explore microbial diversity in the WAP, we characterized bacterial, archaeal, and eukaryotic microbial community composition from two depths (near surface and below the euphotic zone) at four sites representing inshore and offshore conditions in the northern and southern WAP during austral summer, as well as a single inshore sample during winter. We detected differences in bacterial community composition and greater bacterial and eukaryotic richness with depth at the four sites. PCR-amplification failed to enrich archaeal targets in summer surface waters but did detect populations in summer deep and winter surface water. We hypothesize that dissolved organic matter (DOM) input from phytoplankton blooms drives differences in microbial community composition and diversity between depths or seasons. Additionally, sea ice retreat in the spring releases phytoplankton, bacteria and DOM, potentially triggering phytoplankton blooms and altering bacterial community composition and activity. To examine these factors, we conducted two mesocosm experiments in the austral summer, in which we added either small amounts of filtered or unfiltered melted sea ice to 100-m water or DOM exudates (harvested from 20L of Thalassiosira wiessflogii culture) to 10-m and 100-m water. Addition of unfiltered melted sea ice caused increases in phytoplankton abundance and biomass and an early peak in bacterial production, suggesting that water column seeding may trigger phytoplankton growth and bacterial production, while filtered melted sea-ice showed little effect. Direct DOM additions had a dramatic impact on bacterial abundance and production in 100-m water but little effect in 10-m water, suggesting that different factors shape bacterial community dynamics at different depths.

324A High viral impact on picoeukaryotic phytoplankton despite phosphate limitation Douwe Maat*, Corina Brussaard Royal Netherlands Institute for Sea Research (NIOZ), Netherlands

Global climate change predicts strengthened vertical stratification and consequently a shift towards smaller-sized phytoplankton because of enhanced nutrient limitation. Micromonas pusilla, a widespread picoeukaryote from the class Prasinophyceae, is theoretically adapted to cope with low phosphate concentrations. It is known to be readily infected by viruses, affecting its proliferative success, possibly to an extent where bloom formation of this species is suppressed. Here we present for the first time that the negative effect of phosphate limitation on virus replication and production can be overcome by the potential of M. pusilla to utilize organic phosphorus during the infection cycle. Axenic cultures of M. pusilla were pre-grown in chemostats under phosphate limitation. At steady state the cells were infected with viruses and studied over time under batch conditions. However, to maintain the same phosphate input as under chemostat conditions, the infected and non-infected control cultures were spiked with phosphate. We added either inorganic phosphate, simulating the naturally continuous and essential remineralization of inorganic phosphate by the microbial food web, or organic phosphate, simulating the direct uptake of dissolved organic phosphorus deriving from biological activity. Phosphate replete (eutrophic conditions) and starved (severely depleted conditions) cultures were taken along for comparison. A clear negative trend from phosphate replete to phosphate limited to phosphate starved cultures was observed for viral burst size. Besides, the viral latent period was strongly correlated to the severeness of the phosphate limitation. Interestingly, organic phosphate could be readily utilized by the algal host (all three organic forms of phosphate tested) and showed comparable results for the viral growth cycle as inorganic phosphate spiking. Moreover, virus-free lysate as a source of organic phosphate also stimulated the virus production as compared to the starved controls. The consequences of our findings for ecosystem functioning will be discussed taking into consideration the predicted increased occurrence of phosphate limiting ocean systems due to global climate change.

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325A Bacterial hydrolysis of marine particles: identifying temporal breakdown patterns and species-specific carbon substrate assimilation using stable isotope labeling, microarrays and nanosims Xavier Mayali*, Benjamin J. Stewart, Jennifer Pett-Ridge, Peter K. Weber Lawrence Livermore National Laboratory, USA

One of the primary pathways of carbon burial into the deep ocean is the sinking of algal detritus from surface waters. It is well established that bacteria attached to these particles influence the rate of this carbon flux through enzymatic breakdown. While the attached microorganisms can be identified through 16S sequencing, their corresponding biogeochemical functions are not well established. We are studying the patterns of particle breakdown and bacterial assimilation of detrital carbon using a model system of freeze-thawed diatom cultures incubated with natural microbial communities in laboratory microcosms.

Diatom cultures were labeled with 13C, freeze-thawed, and incubated for two weeks with a natural microbial community collected from the surface ocean. Laboratory incubated particles were collected at 4 time points, fractionated into major molecular components (proteins, lipids, polysaccharides) and analyzed for carbon content and 13C isotopic enrichment with isotope ratio mass spectrometry. This enables measurements of carbon flux into and out of the particles, mediated by bacterial attachment and hydrolysis, respectively. Concurrently, non-labeled particles and their attached bacteria were incubated with 13C-labeled carbon substrates (proteins, lipids, and polysaccharides) to identify which bacterial taxa incorporated these substrates into their biomass, using the Chip-SIP (stable isotope probing) method. After 16S sequencing to identify attached microbial taxa, custom phylogenetic microarrays were designed to target these microorganisms, and extracted RNA from the incubations was hybridized to the microarrays. The hybridized probe spots were analyzed for isotopic signal using imaging mass spectrometry with a Cameca NanoSIMS 50, enabling relative quantification of substrate assimilation by distinct microbial ribotypes.

The two methods provide complementary data about carbon fluxes out of the particles and among its molecular components as well as the identity of the microorganisms mediating these changes over time. Our results from these experiments provide evidence of successional shifts and niche differentiation mediated by substrate preference.

326A The impact of enhanced stratification on the structuring of marine food webs Kristina Mojica*, Corina Brussaard Netherlands Institute for Sea Research (NIOZ), Netherlands

Previsioned reinforcements in the vertical stratification, due to global warming, will impact the phytoplankton growth and species composition with potential cascading effects on ecosystem functioning and biogeochemical fluxes. However, our understanding of marine biogeochemical fluxes is currently restricted by a lack of quantitative estimates for loss rates due to viral activity. Until this deficit is filled, the predictive power for the repercussions that global warming will have on these fundamental processes is limited. Combining data obtained from a summer (July-August 2009) field campaign which traversed a north-south stratification gradient in the northeast Atlantic Ocean, we reconstruct the microbial food web budget in order to understand how stratification affects marine food web structure and shapes marine biogeochemical cycling. During this period of the year, the Northeast Atlantic is at its highest level of stratification; however, density profiles showed a latitudinal gradient leading to southern stations having a characteristic deep-chlorophyll maximum (i.e. 0.3 -0.5 µg Chlorophyll a located around 80-100m) which decreased in depth to a surface maximum at the northern most stations. In the south, 26% of the total photosynthetically fixed carbon was released into the dissolved organic matter pool by viral activity and 21% made available to higher trophic levels by grazers, compared to 6% and 20% in northern stations, respectively. These results indicate increased activity of viruses on the mortality of phytoplankton under vertical stratification, allowing sustained activity in stratified regions as viruses are efficient recyclers of biogeochemical elements. The total available carbon (standing stock + production) in the bacterial pool was higher in the north (8.5 x 10-7 µgC L-1) compared to the south (1.1 x 10-7 µgC L-1). In the secondary component, grazing was the dominate loss factor, independent of the region, with 6.1 and 14.1 µC L-1 d-1 accessible to higher trophic levels in the south and north, respectively; compared to 2.5 and 4.3 µC L-1 d-1 being recycled back into the dissolved organic matter pool by the lytic viruses. The Northeastern Atlantic is a key

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region in global ocean circulation, a large sink for atmospheric carbon dioxide and considered to be particularly susceptible to global climate change. Our quantification of the extent to which viruses and grazers contribute to the biogeochemical fluxes under different degrees of stratification has important implications for our understanding of the global oceanic carbon cycle and ecosystem functioning in a changing ocean.

327A Natural ammonium oxidizers' response to chemical changes in the permanent and seasonal oxygen minimum zones of Chile Veronica Molina*1, Camila Fernández2, Hector Levipan3, Osvaldo Ulloa3, Tage Dalsgaard4, Bo Thamdrup5, Niels Peter Revsbech4 1Universidad Andres Bello, Chile, 2CNRS UMR-7621 Banyuls-Sur-Mer, France, 3Universidad de Concepción Chile, 4Aarhus University Denmark, 5University of Southern Denmark Denmark

Oxygen minimum zones (OMZs) off Chile are characterized by suboxic and anoxic conditions 5 - 0.05 μM as a permanent and seasonal feature, off northen and off central-south Chile, respectively. An intense nitrogen cycling is developed in these OMZs driven by reductive dissimilative pathways, such as denitrification, and also by oxidative metabolisms, like the aerobic and anaerobic oxidation of ammonium. Ammonium oxidizers cohabit in the upper boundary of the OMZ competing for ammonium at oxygen deficient conditions. Herein, we evaluate the response of natural ammonia oxidizing archaea and bacteria to chemical changes in the water column of permanent and seasonal OMZs through the in situ quantification of ammonia monooxygenase A subunit transcripts (amoA). Also, the response of archaeal and bacterial amoA transcripts and anammox metabolism were followed in oxygen controlled bioreactor experiments only off northern Chile. In the permanent OMZ, archaeal amoA transcripts were dominant compared to bacterial amoA. A similar expression, up to 80 copies/ng RNA, were observed in the oxic layer and upper boundary of the OMZ, despite contrasting oxygen, i.e., 50 versus <5 μM, and ammonium, i.e, 0.4 versus 0.01 μM concentrations, respectively. Oxygen controlled bioreactor experiments carried out with water coming from the core of the oxygen minimum zone showed a quick archaeal amoA transcript response to oxygen and ammonium additions, with a consistently higher activity in the presence of both elements. Also, the co-activity of aerobic and anaerobic ammonia oxidizers was observed at <2 μM oxygen conditions. In the seasonal OMZ, also archaeal amoA transcripts were dominant and higher within the oxyclines (60-430 copies/ng RNA) compared with surface or bottom layers of the water columns, mainly during the development of the OMZ in the upwelling period (spring-summertime). In the seasonal OMZ in situ archaeal amoA transcripts were significantly positively correlated with in-situ nitrate concentrations, but not with oxygen or ammonium. In total, these results indicate that ammonium oxidizers, particularly archaeal ones, are a versatile group within the realm of the oxygen minimum zones, able to cope with low substrate conditions and main drivers of nitrification. Their rapid responses to ammonium and oxygen experimental additions indicate that oxyclines are perfect habitat for this prominent microbial group within expanding oxygen minimum zones.

328A Metabolic versatility of dominant planktonic sulfur-oxidizing γ-Proteobacteria in a costal oxygen minimum zone Alejandro Murillo*1, Salvador Ramírez-Flandes1, Edward DeLong2, Osvaldo Ulloa1 1Universidad de Concepción, Chile, 2Massachusetts Institute of Technology, USA

Marine oxygen minimum zones (OMZs) are prominent in the eastern Pacific Ocean, the Arabian Sea, and the southeast Atlantic Ocean off West Africa, and are believed to be expanding. Also, anoxic/euxinic systems develop in enclosed seas, such as the Black Sea and the Baltic Sea, or in isolated basins like the Cariaco Basin and Saanich Inlet. In all of these oxygen-deficient systems, chemolithoautotrophic Epsilon- and Gammaproteobacteria appear to play a central role in the cycling of carbon, nitrogen, and sulfur species. Specifically, the SUP05 linage of the Gammaproteobacteria, which is related to sulfur-oxidizing symbionts of deep-sea clams, has been implicated in a “cryptic sulfur cycle” in the permanent OMZ of the eastern South Pacific (ESP), off the coast of northern Chile. A previous metagenomic analysis of members of the uncultured SUP05 lineage in the Canadian fjord Saanich Inlet has shown them to be metabolically versatile. For example, they have the genetic potential for sulfur oxidation coupled to nitrate reduction, with the concomitant production of the greenhouse gas nitrous oxide (N2O). Based on this metagenomic analysis, however, SUP05 bacteria seem to be obligate chemolithoautotrophs that are incapable of aerobic respiration. Here we present results of a novel metagenomic analysis of SUP05-like microorganisms from a coastal upwelling ecosystem in the subtropical ESP. This ecosystem experiences seasonal anoxia and accumulation of

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nitrite and ammonium at depth, with corresponding increases in members of the SUP05 lineage. The ESP SUP05 members have a significantly broader gene complement than the Saanich Inlet SUP05 group. Analyses of de novo assembled contigs indicated the presence of a complete aerobic respiratory complex based on the cytocrome bc1 oxidase, which could provide them the capability of aerobic respiration when oxygen is present. Also, apart from the RuBisCO operon, they appear to encode the complete TCA cycle and several transporters for dissolved organic carbon species, suggesting a mixotrophic life-style. Finally, the ESP SUP05 group also has the genetic potential for reducing nitrate to ammonium. Thus, the success and extended distribution of sulfur-oxidizing bacteria in oxygen-deficient marine ecosystems appears due not only to their previously recognized anaerobic metabolic versatility, but also to their capacity to function under aerobic conditions using different carbon sources.

329A Plastic pollution and marine microbes: Identifying the plastic colonising community in the ocean Sonja Oberbeckmann*, A. Mark Osborn University of Hull, United Kingdom

Since the mass production of plastic commenced 100 years ago, plastic particles have been accumulating in the marine environment. Only recently the alarming scale of plastic pollution in the world’s oceans has been recognized, in particular through the discovery of the Pacific and Atlantic Garbage Patches. Plastic particles can enter the marine food web and affect organisms at all levels including microorganisms. Microorganisms, such as bacteria and microalgae, are known to adapt very quickly to newly emerging niches and might therefore be able to use plastic particles as habitat. However, it still has to be clarified, which microorganisms are colonising plastic in the oceans, and what are the possible consequences of this colonisation. Our study is approaching these questions by investigating microbial plastic colonisation in waters around the UK. We deployed polyethylene terephthalate bottles for 6 weeks at jetties and buoys in the North Sea, in estuarine and marine conditions. Besides this controlled approach, we performed Manta trawl sampling along a transect in the North Sea, the English Channel, the Celtic Sea and the Bristol Channel, in order to collect plastic pieces from the environment. Plastic samples from both the in situ exposure experiment and directly collected from the ocean were checked visually for microbial colonisation using fluorescence microscopy. We managed to extract DNA from the plastic pieces and to amplify 18S as well as bacterial and archaeal 16S rRNA gene fragments. To provide information about the diversity of microbial plastic colonisers, community fingerprinting (DGGE) was carried out. First results reveal differences in diversity and composition of the plastic colonising communities of both, eukaryotes and prokaryotes, regarding sampling station. The microbial community attached to plastic in estuarine waters shows a higher diversity and variance compared to the one in marine waters. However, certain microbial eukaryotes and bacteria seem to be attached to plastic at every location. Currently, prominent DNA fragments eluted from DGGE gels are being sequenced in order to identify microbial plastic colonisers. Next generation sequencing of chosen samples will give more information on the function of these colonisers. This work represents a significant step towards the understanding of microbial colonisation of plastic and will help to identify its possible positive and negative consequences.

330A Transcrioptome Analysis of Light-utilizing Strain Candidatus Puniceispirillum marinum IMCC1322, a Member of SAR116 Group Bacteria Hyun-Myung Oh*1, Kaekyoung Kwon1, Ahyoung Choi2, Dongmin Kang2, Yong Hak Kim3, Jang-Cheon Cho2 1Korea Ocean Research & Development Institute, South Korea, 2Inha University, South Korea, 3Catholic University of Daegu, South Korea

The purpose of this study is to elucidate the proteorhodopsin function based on the genome of strain IMCC1322 which is a cultivated species of SAR116 group from East Sea of Korea. Light-enhanced photoheterotrophy of strain IMCC1322, whose phenotypic characteristics were revealed by cultivation and polyphasic studies, was interrogated by the transcriptomics. Samples of mRNA of dark/light/diel IMCC1322 cultures were obtained at days 3 and 6 post inoculation. MICROBExpress™ Bacterial mRNA Enrichment Kit and Illumina Genome Analyzer was used and the obtained sequence reads were mapped to the IMCC1322 genome (GenBank Acc. No. CP001751) using Burrows-Wheeler Aligner (BWA) (K-mer=32, mismatch=2). Filtered and aligned reads were counted using BEDTools (version 2.9) and were summarized in BAM file formats. Here we report the mRNA profiling of

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IMCC1322 which might be the first attempt to the biogeochemistry of light-utilizing SAR116 group in the ocean and the transcriptomes revealed that green light (max. wavelength =522nm) drives respiratory metabolism and cessation of bacterial aging of IMCC1322 after stationary phase.

331A Effect of Oxygen Minimum Zone formation on communities of marine protists William Orsi*1, Young Song2, Alyse Hawley2, Monica Torres Beltran2, Steven Hallam2, Virginia Edgcomb1 1Woods Hole Oceanographic Institution, USA, 2University of British Columbia, Canada

Changes in ocean temperature and circulation patterns compounded by human activities are leading to oxygen minimum zone expansion with concomitant alteration in nutrient and climate active trace gas cycling. Here, we report the response of microbial eukaryote populations to seasonal changes in water column oxygen-deficiency over a 4-year time series using Saanich Inlet, a seasonally anoxic fjord on the coast of Vancouver Island British Columbia, as a model ecosystem. We combine 454 pyrosequencing and Sanger sequencing approaches with multivariate statistical methods to reveal shifts in operational taxonomic units during successive stages of seasonal stratification and renewal. A meta-analysis is used to identify common and unique patterns of community composition between Saanich Inlet and the anoxic/sulfidic Cariaco Basin (Venezuela) and Framvaren Fjord (Norway) to show shared and unique responses of microbial eukaryotes to oxygen and sulfide in these three environments. Our analyses also reveal temporal fluctuations in rare populations of microbial eukaryotes, particularly anaerobic ciliates, that may be of significant importance to the biogeochemical cycling of methane in oxygen minimum zones.

332A Fungal diversity associated with Brazilian marine sponge Dragmacidon reticulata Michel Passarini*1, Lara Sette2 1Unicamp - Campinas State University, Brazil, 2Department of Biochemistry and Microbiology - UNESP, Brazil

Recently the research concerning marine microorganisms has increased but little is still known about the diversity of fungal communities associated with marine sponges. Studies on microbial diversity by using microscopy and cell counts have shown we can recover up to 10% of the microorganisms from environmental samples through the isolation and culturing. In this context, the aim of the present study was to investigate the diversity of fungi from marine sponge D. reticulata by culture-independent approaches, including DGGE and direct ITS-rDNA sequencing. The sponge samples collected at different places were crushed and the total community DNA was extracted with PowerSoil® DNA Isolation kit. The DNA was amplified with the set primers ITS1F/ITS4 and ITS3/ITS4-GC (nested PCR). The amplicons generated with primers ITS3/ITS4-GC were put into a 6% polyacrylamide gel with a denaturing gradient of 20-55% and the fragments were separated by 16 hours at 100 V at 60 ºC. Bands were stabbed from DGGE gel, re-amplified without clamp GC and cloned into E. coli. For ITS-rDNA direct sequencing was used the set of primers ITS1F/ITS4. The positive clones were amplified with primers M13F/M13R and sequenced into automatic sequencer ABI 1500 for both strategies. Sequences were compared with ITS-rDNA sequences data from the public GenBank using BLASTN. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 4.0 and the diversity was estimated using Mothur program. Clones representatives of eight different genera were identified from the samples DR6 and DR9, one from phylum Basidiomycota: Ganoderma (2 from DR9) and seven from phylum Ascomycota: Aureobasidium (18 clones from DR9), Devriesia (19 from DR6), Fusarium (9 from DR6), Metschnikowia (1 from DR9), Microsphaeropsis (14 from DR9 and 23 from DR6), Penicillium (20 from DR9), Preussia (1 from DR9), being one of the clones from DR9 classified as uncultured fungal clone. Fungal diversity associated with DR9 seems to be higher than the one achieved in DR6. The genera Aureobasidium, Dreviesia, Metschnikowia and Preussia were only found using culture-independent strategy. In a previous study using culture-dependent strategy and the same samples (data not shown), twenty other genera could be identified. These results suggested that both strategies need to be done in order to achieve a more accurate diversity from environmental samples. In addition, the fungal diversity from samples DR6 and DR9 indicates that marine ecosystems may represent a rich source for the discovery of new natural products.

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333A Molecular and culturable analysis of microbial diversity present in highly-copper-contaminated Chilean marine sediments Laurent Quillet*1, Besaury Ludovic1, Marty Florence2, Buquet Sylvaine1, Muyzer Gerald2 1UMR CNRS 6143 Université de Rouen, France, 2University of Delft, Netherlands

Marine sediments are considered as sink for heavy metals and in those ecosystems, copper is described as an important pollutant due to its toxicity and his potential to be accumulated though the food chain.

Cores of 25 cm were taken from two Chilean marine sediments in the north of Chile contaminated by a channel from a copper-mine. The first site is named Palito and is highly-contaminated (800ppm); the second one is named Embouchure and is less contaminated (300ppm). Cores were cut in sections of 2 cm to study culturable diversity in oxic and anoxic conditions. For the anaerobic culturable microorganisms, research particularly focused on sulfate-reducing (SRB) and thiosulfate-reducing bacteria because sulfide production can lead to the formation of insoluble metal sulfides which reduce the bioavalibility of metal ions in ecosystems. Investigation of iron-reducing bacteria was also realised because iron oxides can adsorb several heavy-metals.

In oxic conditions, main group of strains found was part of the genus of Bacillus. 24 isolates on the 43 isolates from genus Bacillus presented a resistance to copper between 300 ppm and 400ppm and 2 isolates had a resistance superior to 400ppm. The rest of isolates (n=22) belonged to five minority genus: Acinetobacter, Pseudomonas, Shewanella, Burkholderia, Arthrobacter; 11 isolates exhibiting a resistance superior to 200 ppm.

In anoxic conditions, 11 strains of SRB were isolated and were all from genus Desulfovibrio. Resistance to copper was tested for those strains and 6 exhibited a resistance superior to 200 ppm. The others strains isolated were only fermentative bacteria and belonged to 4 genus: Virgibacillus, Alkalibacterium, Sphingobacterium, Bacillus. Iron-reducing bacteria were searched and only one strains was isolated belonging to the genus Desulfuromonas (such strain will be described soon as new species). This strain present a very important resistance to copper which can reach more than 1000 ppm.

Presence of copper-resistance genes copA, cusA and pcoA was tested on DNA extracted from the 82 strains isolated; cusA and pcoA genes were not detected and gene copA was only detected five times, majoritary for SRB strains suggesting for those strains a better efficiency expulsion of ATP-ase pump to detoxify cells in highly contaminated sediments. Molecular analysis by clone libraries of 16SrDNA revealed that majoritary clones are found for Palito site for Actinobacteria, Gammaproteobacteria and Planctomyces. For Embouchure site, majoritary clones were found for Alphaproteobacteria, Gammaproteobacteria and Bacteroidetes but no relationship was found between majoritary groups described with molecular and culturable methods.

334A 454 pyrosequencing analysis of microbial communities and qPCR detection of cyanotoxin genes in a marine system in Singapore Damien Raingeard1, Charmaine Ng*1, Peter Steinberg2, Ole Larsen3, Diane McDougald4 1Advanced Environmental Biotechnology Centre, Nanyang Environment and Research Water Institute, Nanyang Technological University, Singapore, 2Sydney Institute of Marine Science, Chowder Bay, NSW, Australia, 34DHI-NTU Water & Environment Research Centre and Education Hub, Nanyang Technological University, Singapore, 4Centre for Marine Biofouling and Bio-Innovation, School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia

Singapore is the busiest container port with one of the highest population densities in the world. Transhipment, vessel and ballast water discharges, wastewater effluent inputs, runoff waters and land reclamation activities are factors contributing to eutrophication and anthropic pressure on coastal marine ecosystems. The aim of this study was to link seasonal changes to shifts in microbial communities using 454 pyrosequencing technology, targeting the16S rRNA gene V3 hypervariable region. The sampling site was located at Raffles marina (1°20.53’N 103°38.22’E), at 0.2 nautical miles off the Malaysian borderline and next to the heavy industrial area of Singapore. Over a period of one year, fortnightly water samples of 25 litres were collected at low tide, between the surface and a 2

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meter depth. Biomass was sequentially filtered through 25, 3 and 0.1 µm membranes. For each sample, ~3000 reads were analysed using Mothur and the Ribosomal Database Project (RDP). Presence and abundance of cyanotoxins (saxitoxin, cylindrospermopsin, nodularin and microcystin) were also assessed using real time PCR. Over the sampling period, for the major phyla identified (Proteobacteria, Cyanobacteria, and Bacteroidetes), significant subtaxa shifts were observed within Alpha-, and Beta- or Gammaproteobacteria, Cyanobacteria and Chloroplast genera, and Flavobacteria and unclassified classes. At lower taxonomic ranks, data analysis showed complex seasonal operational taxonomic units (OTUs) variations over the Northeast, Southwest and inter-monsoon periods. These data were further used to design probes to be incorporated into an aquatic microbial early warning system to predict toxic blooms of phytoplankton in Singaporean marine environments.

335A Comparative Genomics within a Coastal SAR11 Population Michael Rappe*, Jana Grote, Megan Huggett University of Hawaii, USA

Since their initial discovery in 1990, a variety of molecular biology and microscopy techniques have revealed that a monophyletic lineage of free-living marine bacteria known as SAR11 comprises one of the most dominant groups of organisms on Earth, where they typically account for 25% or more of the prokaryotic cells in seawater of the global ocean. Due to its high abundance and ubiquitous distribution, SAR11 remains the focus of much scientific attention. Contemporary thought is that the study of SAR11 microorganisms is vital to understanding the ecology of planktonic marine systems, including organic carbon and nutrient cycling in the global ocean. This diverse lineage (identities as low as 82% in the most highly divergent 16S rRNA gene sequences) encompasses at least five major, basal subclades that can be distinguished by their spatial and/or temporal patterns of distribution. However, until recently only a handful of closely related SAR11 strains had been brought in to culture in the laboratory for controlled experimentation and interrogation. Using modified dilution to extinction culture techniques, we isolated 53 axenic SAR11 strains from coastal seawater of Oahu, Hawaii, in the tropical Pacific Ocean. In order to begin to quantitatively investigate how populations of bacteria such as SAR11 have evolved, we have determined whole genome sequences of seven strains to date, including isolates spanning four of the major SAR11 lineages - four from SAR11 subgroup Ia and one each from SAR11 subgroups II, III, and V. Consistent with previous published observations, all of the SAR11 strains analyzed here possess small (1.24-1.44 Mbp) genomes of low GC (28.6-32.3%) content. The four closely related genomes of SAR11 subgroup Ia (>98.5% 16S rRNA gene sequence similarity) shared between 83.3-88.3% of their gene content in pair-wise comparisons, with an average of 79.4% of total genes shared between all four strains. Thus, this initial glimpse revealed significant variability within the genomes of a single SAR11 population, including the variable presence of genes with predicted functions of biogeochemical relevance. The remaining SAR11 genomes are each members of phylogenetically distant lineages, and provide insight into potential physiological traits that distinguish these subgroups. Improvements in sequencing technologies leading to increased throughput and decreased cost now provide the opportunity to sequence and compare hundreds of genomes at once, providing unprecedented opportunities to characterize and quantify population-level genome dynamics in environmentally relevant microorganisms.

336A Ocean acidification affects benthic microbial communities along natural pCO2 gradients associated with shallow tropical gas vents Felix Raulf*1, Katharina Fabricius2, Sven Uthicke2, Dirk de Beer1, Raeid Abed3, Antje Boetius1, Alban Ramette1 1Max Planck Institute for Marine Microbiology, Germany, 2Australian Institute of Marine Science, Australia, 3Sultan Qaboos University, Oman

Naturally occurring CO2 venting systems mimic conditions that resemble intermediate to high pCO2 levels as predicted for our future oceans. They thus offer a unique opportunity to investigate long-term effects of ocean acidification on marine microbial communities. We examined the composition and diversity of benthic bacterial and archaeal communities along natural pCO2 gradients in the range of IPCC projections for the next century (400 to 1000 µatm), located at Milne bay, Papua New Guinea. At those sites the emitted volcanic gas contains more than 99% CO2, CO2 emissions have most probably been stable for several decades and the effect of CO2 is not confounded by temperature fluctuations. Bacterial community structure was analyzed by community fingerprinting (Automated Ribosomal Intergenic Spacer Analysis, ARISA) and by 454 pyrosequencing of 16S rRNA genes. All data were further analyzed together with environmental parameters including pH, pCO2, carbon and nitrogen

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content, in a multivariate statistical approach. The number of bacterial ARISA operational taxonomic units was negatively correlated to increasing CO2 concentrations and microbial types found at low impacted areas, close to ambient levels, were significantly different from those identified at sites with elevated CO2 conditions. Changes in the community beta-diversity were characterized by increasing heterogeneity with increasing pCO2. More in-depth analysis of bacterial as well as archaeal communities, based on 454 pyrosequencing data, and their association with contextual environmental parameters will be presented and discussed. This study clearly demonstrates how studying microbial diversity under natural conditions of high pCO2 levels provides novel insights on the impact of ocean acidification on microbial communities.

337A A microcosm study on oil degrading bacteria of the Archipelago Sea in South-West Finland Anna Reunamo*1, Lasse Riemann2, Piia Leskinen3, Kirsten S. Jørgensen1 1Finnish Environment Institute, Finland, 2University of Copenhagen, Denmark, 3University of Turku, Finland

The aim of our study was to test the natural petroleum hydrocarbon degrading capacity of the brackish Baltic Sea. We wanted to test whether there is a difference between oil degradation potential of polluted and pristine sites and how quickly the microbial community responds to new oil exposure in unpolluted sites, compared to sites with a chronic oil exposure.

A microcosm experiment was carried on using diesel-spiked sea water and control microcosms without diesel addition. Sea water for the experiment was taken from one pristine and two oil-exposed sites. Bacterial community fingerprinting was done using terminal restriction fragment length polymorphism (T-RFLP) and samples from selected microcosms were sequenced. In addition, quantitative PCR was carried out to detect and quantify PAH-degradation genes. Degradation of oil was monitored during the experiment by heptane extraction and gas chromatograph equipped with a flame ionization detector.

Bacterial communities in diesel exposed microcosms diverged from the non-exposed control microcosms in the course of the experiment. The dominating bacterial groups after 14 day diesel exposure were different depending on the sampling site. In comparison to oceans, gammaproteobacteria were not the dominating group in diesel-spiked microcosms. Instead, betaproteobacteria were most abundant in microcosms originating in polluted, and actinobacteria in microcosms in pristine sites. Diesel was degraded in all of the microcosms during the 21 day experiment; the remaining portion varied from 30% to 60% and was not dependent on the sampling site. PAH-degradation genes were detected from all of the microcosm samples and the initial sea water studied. The number of gram-positive PAH-degrading genes remained rather constant during the experiment, but there was a noticeable shift in gram-negative PAH-degrading genes.

Hydrocarbon degrading bacteria, diesel degradation and PAH-degradation genes were detected in the microcosm experiment. However, the bacterial groups in the Baltic Sea differ from the ones in oceans. The community structure of the hydrocarbon degrading bacteria seems to be different in pristine and oil exposed sites. The previous pollution level did not affect the degradation rate in our experiment.

338A Generalist bacteria colonizing the water masses of the amundsen sea polynya Inga Richert*1, Julie Dinasquet2, Ramiro Logares3, Lasse Riemann2, Annelie Wendeberg4, Bertilsson Stefan5 1Uppsala University, Sweden, 2Marine Biological Section, University of Copenhagen, Denmark, 3Institute of Marine Sciences, CSIC, Spain, 4Helmholtz Centre for Environmental Research – UFZ, Germany, 5Uppsala University, Department of Ecology & Genetics, Limnology, Sweden

Here we present a spatial survey of the Amundsen Sea microbiota using regrowth experiments to identify the mechanisms that explain biogeography. The Amundsen Sea, located in the Southern Ocean, represents a unique habitat, exhibiting high and seasonally variable productivity. The reoccurring retreat of the ice pack during the austral summer leads to high UV penetration in the water column and high primary production by patchy phaeocystis blooms.

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We sampled the bacterioplankton communities in 15 stations, and in each station, the different water masses. The turnover and growth of prokaryotic populations was followed in 7-day regrowth experiments without grazers. From three sites within the Amundsen Sea Polynya sterile filtered seawater was inoculated with 0.65 µm filtered seawater from either surface or deeper water masses. Each treatment was done in triplicates under light and dark condition to additionally assess the effect of light on population growth. Samples for later 454-pyrosequencing of 16S rRNA amplicons were taken from the initial inoculum and from the individual populations after 7 days of growth.

We observed a significant clustering of the microbial communities according to the water masses and the distance to ice packs as well as continental ice shelfs. Across all water masses, the bacterial communities were dominated by Flavobacteria (Polaribacter), Gammaproteobacteria (Oceanospirillaceae, Piscirickettiaceae) and Alphaproteobacteria (Pelagibacter, Sulfitobacter). After 7 days of incubation in the dilution cultures, community composition was not strongly affected by the removal of grazers. In particular, no recruitment of initially rare opportunistic species was observed. Instead, the inoculum source had a strong effect in diversity and species richness. Contrary to our expectations, light hampered microbial growth and less diverse communities, dominated by a few, likely photoresistant or phototrophic populations, proliferated in the regrowth cultures exposed to light.

Comparisons between natural populations from the sampled stations with those proliferating in the regrowth experiments indicate that the summer microbiota of the Amundsen Sea Polynya is dominated by groups that are apparently good competitors for nutrients and carbon. We suggest that such resource driven structuring of the microbial community is prominent in the constantly changing environmental conditions typical waters of Polynyas in the Southern Ocean.

339A Strongly stratified functional capacities of microbial communities in the Landsort Deep, Baltic Sea Sara Sjöling*, Petter Thureborn Sodertorn University, Sweden

Upon eutrophication the gradually increasing areas of hypoxic and anoxic deep waters and sediments of the Baltic Proper result in a steep oxygen gradient. This is particularly evident at the Landsort Deep. Given the wide range of environmental conditions, spanning hyposaline surface waters through anoxic sediment with significant accumulation of heavy metals, we sought to establish whether the microbial communities through the water column and into the sediment were taxonomically distinct from one another, and whether each exhibits distinct metabolic capacity. For this purpose we applied direct metagenomic sequencing of total DNA extracts of samples from three depths of water (10, 75, 400 m) and sediment (466 m) at Landsort Deep. Functional capacity and taxonomic profiles were compared using multivariate statistical analysis, for example correspondence analysis and non- metric multidimensional scaling, of annotated sequence data. Results reveal clear stratification of the communities along the depth transect, most marked for predicted gene function but also for taxa (family). The stratification was obvious in both carbon and nitrogen transformation processes. For example, the potentially ammonium oxidising Thaumarchaea was frequent in the 75 m and 400 m samples, indicating an ecological importance of archaeal nitrification and ammonia oxidation below hypoxic depths. We also compared Landsort Deep metagenomes to other aquatic metagenomes through large-scale comparative network analysis (implemented through MEGAN ). The only communities that were similar to the 400 m and sediment communities were those of the Marmara Sea, which also has a strongly stratified water column, while the surface water community was more similar to surface water communities of the Western Channel. The deeper communities of the Landsort Deep were enriched in genetic prerequisites for a life-style adapted to attachment and utilization of organic material for example chemotaxis, pilus, quorum sensing, biofilm formation, degradation of polysaccharides and aminosugars. These communities also had an overrepresentation of cadmium and zinc resistance genes. This suggests a copitrophic community reflecting both eutrophication and the historical input of environmental pollutants into the Baltic Sea.

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340A Isolation of anaerobic nitrogen fixers from sediments of Narragansett Bay, RI, USA Rodrigue Spinette*, Shelley Brown, Annaliese Jones, Bethany Jenkins University of Rhode Island, USA

Measurements of nitrogen (N) flux from the sediments of several estuaries, including those of Narragansett Bay, have recently indicated that N fixation might occur episodically at significant rates (up to 80 g N m2 yr-1). Using degenerate primers targeting nifH, a gene encoding an essential subunit of the N fixation enzyme nitrogenase, and by following nifH gene expression, we have shown that anaerobic N fixing microbes are widely distributed and seasonally active throughout Narragansett Bay benthic sediments. The active diazotrophs in the bay are predominantly in two phylogenetically distinct groups of organisms: the first most abundant group containing the geobacteraceae and the second most abundant the desulfovibrionaceae. The combined molecular and biogeochemical data suggests that the N cycle within estuarine sediments may be impacted by N fixation on temporal and geographical scales that remain unresolved. Furthermore, the impact of combined N on the diazotrophy of these anaerobes is entirely unknown. Our aim is to use cultivation-based methods to examine the importance of several factors on the control of N fixation in diazotrophs living within estuarine sediments, including the availability of different electron acceptors and of combined N sources. Three types of enrichment cultures have been generated using sediment inocula from Narragansett Bay. These cultures include sterile sediment from the bay and liquid medium lacking N but containing one of three electron acceptors: sulfate, elemental sulfur, or iron (III). Using RT PCR, we have confirmed that all three types of enrichment cultures express nifH. We have also measured significant rates of N fixation for the cultures containing sulfate using labeled 30N2 gas incubations and isotopic analysis. Using an anaerobic roll tube method and media containing select electron acceptors, we are also isolating organisms representative of the two dominant groups among the N fixers found in Narragansett Bay. From the parent enrichment cultures containing sulfate, two members of the desulfovibrionaceae have successfully been isolated. Growth rates are essentially the same for one of the isolates even when combined N in the form of ammonia or nitrate is added. While isolation of N fixers in the geobacteraceae has proven more difficult compared to the desulfovibrionaceae, isolation efforts with further refinements in the media used for enrichment and isolation show promise. Ultimately, the use of these enrichment cultures and bacterial isolates in combination with N fixation rate measurements will helps us identify the factors that drive N fixation in the environment.

341A Assessing copper stress response in natural communities of marine Synechococcus Rhona Stuart*1, Kristen Buck2, Randelle Bundy1, Bianca Brahamsha1, Brian Palenik1 1Scripps Institute of Oceanography, UC San Diego, USA, 2Bermuda Institute of Ocean Sciences, Bermuda

Marine picocyanobacteria including Synechococcus and its sister taxa Prochlorococcus are a highly diverse group that are found throughout the world oceans and contribute significantly to primary production and carbon cycling. Copper can be both a micronutrient and a toxicant and of the main phytoplankton groups marine Synechococcus is the most sensitive to copper toxicity. However, copper stress response in marine Synechococcus is not well understood and there is no method for detecting copper stress in natural populations. We looked at copper stress response in natural populations of marine Synechococcus looking at both short-term (2 hour) gene expression response using targeted gene expression microarrays and long-term (14 day) Synechococcus community changes. We designed and tested custom microarrays based on two genomes of cultured strains and found some similarities in the transcriptional copper stress response of this mixed community to that of culture-based marine Synechococcus response. The long-term incubations revealed a shift in Synechococcus diversity in the copper-treated samples, suggesting that there are copper tolerant subpopulations of marine Synechococcus. Together, this data reveals a complex diverse response of the Synechococcus community to copper stress.

342A Effects of light on the proteorhodopsin-containing marine gammaproteobacterium Photobacterium angustum S14 under batch and chemostat conditions Marcelino Suzuki, Alicia Courties*, Philippe Lebaron University Paris 6 - Observatory of Banyuls - LOMIC, France

Over the past decade, the application of genomic and environmental genomic techniques to the study or marine bacterioplankton communities revealed that a large fraction of these organisms -

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responsible for important reactions in the global carbon cycle - are in fact photoheterotrophs. Photoheterotrophs obtain their cellular energy both from the oxidation of organic carbon and from light energy. Among the most ubiquitous and abundant photoheterotrophs are prokaryotes containing proteorhodopsins (PCPs), which are trans-membrane, retinal dependent proton pumps. Despite the large amount of knowledge gathered in the past few years on PCPs, the contribution of light energy in the overall carbon budget of these organisms remains unclear. Studies examining growth rates and yields in batch culture show contrasting results, in one cases showing an effect of light in growth rates and yields, while in others proteorhodopsin phototrophy was shown to improve starvation survival. To the best of our knowledge, to date the effect of light on carbon utilization efficiency by PCPs has not been addressed. We will present the results of studies of batch growth under different light conditions and carbon utilization efficiency by the proteorhodopsin containing marine gammaproteobacterium Photobacterium angustum S14 under different light conditions in photobioreactors and chemostat conditions. The experimental design of these experiments was to maintain a constant dilution (that is growth) rate, while measuring differences in yield during illuminated and dark conditions. The results indicate that even though batch growth was not affected by light we were able to observe a significant positive effect of light on carbon utilization efficiency event at high (albeit limiting) carbon concentrations, high dilution rates (0.1 h-1) and high cell concentrations (108/ml).

343A Ecosystem-specific adaptation of Prochlorococcus, SAR11, and viruses in the Red Sea Luke Thompson*1, Matt Cahill1, Chris Field2, Ali Awami1, Markus Hadwiger1, Ulrich Stingl1 1King Abdullah University of Science and Technology, Saudi Arabia, 2Dalhousie University, Canada

The epipelagic zone of the Red Sea is an oligotrophic marine ecosystem with very high solar irradiation, temperature, and salinity. Historically, very little work has been done to study the microbial community in these waters. However, with the establishment of the King Abdullah University of Science and Technology (KAUST) on the eastern shore of the Red Sea, providing unfettered access throughout the eastern Red Sea, a clearer picture of its microbial community is starting to emerge. In this talk, I will highlight some of the work ongoing in the Red Sea Research Center at KAUST to understand the community structure and evolutionary adaptations of Red Sea bacteria and viruses, particularly the groups Prochlorococcus, SAR11/Pelagibacter, and cyanophage. Work in our center by Ngugi et al. (Molec Ecol, 2011, doi: 10.1111/j.1365-294X.2011.05378.x) has shown that, at the taxonomic level, Red Sea surface waters are not unlike other oligotrophic ecosystems such as the major open-ocean gyres; for example, the bacterial community is dominated by SAR11 and Prochlorococcus. More fine-scale approaches, however, are beginning to reveal significant genomic and geographic diversification of the Red Sea’s microbial populations. On the genomic scale, a metagenomic comparison of the Red Sea with the North Pacific and North Atlantic Oceans and the Mediterranean Sea shows its Prochlorococcus population to be enriched in DNA repair genes and its SAR11 population to be enriched in genes for degrading osmolytes. These suggest evolutionary pressures applied by high light levels and low nitrogen levels, respectively. Single-cell genomics of Prochlorococcus and SAR11 cells reveals genomic features specific for the Red Sea that have not been identified in previously sequenced genomes. On the geographic scale, sampling of 50 depth profiles aboard a KAUST/WHOI expedition spanning the eastern Red Sea from Sinai to Jizan has provided a fine-scale look at community structure in the Red Sea, especially as it relates to key physical and chemical parameters. We will present these results with the aid of the Red Sea Visualizer, a 3D viewer for oceanographic data on the iPad and the Web developed at KAUST. Together, our work shows the microbial community in the sunlit waters of the Red Sea to be not unlike other nutrient-limited seas, but with important adaptations to its high light and particular nutrient conditions. Future work will help unravel adaptations to the Red Sea’s high water temperatures, which will be important for predicting microbial responses to global warming.

344A Genome characterization of bacterial strains isolated from the biofilm of a denitrification reactor treating seawater Richard Villemur*, Céline Villeneuve, Christine Martineau, Florian Mauffrey INRS-Institut Armand-Frappier, Canada

The Montreal Biodome established a methanol-fed, fluidized denitrification reactor for controlling the concentration of nitrate in its three million liter seawater aquarium. The bacterial biota of the denitrifying biofilm in the reactor was estimated to contain between 15 and 20 species. Approximately 80% of the biofilm is composed of bacteria affiliated to the Hyphomicrobium and Methylophaga

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genera. Four bacterial strains have been isolated so far from the biofilm among which three methylotrophic bacteria: the nitrate-reducing Methylophaga nitratireducens JAM1, Methylophaga frappieri JAM7 and the denitrifying Hyphomicrobium nitrativorans NL23. The fourth one is Nitratireductor aquibiodomus, a heterotrophic bacterium. We aim to understand how these bacteria work together in the denitrifying biofilm. By obtaining the complete genome of these strains, predicted metabolic pathways will reveal the mechanisms of action of these strains relative to the denitrifying activities, but also to their symbiotic relationship in the biofilm.

The sequencing of the genome was achieved for strains JAM1 and JAM7, and is undergoing for the two others. The genome of strain JAM1 (3137192 bp) harbors 3043 predicted coding open reading frames (ORFs), of which 2476 have a predicted function. For JAM7 (2697465 bp), 2698 ORFs of which 2194 have a predicted function were found. Strain JAM7 contains also a 47825 kb plasmid. It was shown previous that strain JAM1 can grow under denitrifying conditions by reducing nitrate into nitrite (not further) and this was correlated with the presence of two nitrate reductase narG genes. The strain JAM1 genome sequence confirmed the presence of two nar operons, but interestingly also two nor operons (nitric oxide reductase) and one nos operon (nitrous oxide reductase). In addition, a nirK sequence encoding for an 82 amino acid truncated nitrate reductase was found, which could explain that strain JAM1 can only reduce nitrate into nitrite. No genes involved in denitrification were predicted for strain JAM7.

345A Comparative community proteogenomics of estuarine and coastal ocean ecosystems David Walsh*1, Anna Georges1, Ting Ting Cui1, Heba El Swais1, Susanne Craig2, William Li2 1Concordia University, Canada, 2Bedford Institute of Oceanography, Canada

The chemistry and biology of the ocean is changing at a global scale due to human activity. Rising atmospheric CO2 concentrations and temperature are leading to ocean acidification and a reduction in subsurface dissolved oxygen concentrations. Agricultural and industrial activity is resulting in elevated nutrient and pollutant levels in coastal waters. The St. Lawrence Estuary (SLE) is a massive and productive aquatic ecosystem in eastern Canada. Similar to many aquatic systems, progressive acidification and depletion of dissolved oxygen is on going in SLE bottom waters, making the SLE a dynamic system faced with long term changes in environmental factors. How these changes may impact microbial community structure and biogeochemical processes is difficult to predict since few studies linking microbial ecology and biogeochemistry in the SLE exist. Here we combine 16S rRNA analysis, metagenomics, and tandem mass spectrometry-based shotgun proteomics to explore metabolic diversity of whole microbial communities from the SLE, as well as communities from a nearby seasonally hypoxic coastal basin (Bedford Basin, Nova Scotia). In surface waters, the most frequently identified expressed proteins corresponded to the periplasmic components of ATP-binding cassette (ABC) transporters. These diverse ABC transporter proteins included those specific for amino acids, peptides, sugars, organic acids and inorganic nutrients. During a phytoplankton bloom, the protein detected at highest abundance was methanol dehydrogenase from Beta-proteobacteria, supporting several recent findings that the metabolism of one carbon compounds may be important in the biogeochemistry of the coastal ocean. In the seasonally hypoxic Bedford Basin, we detected a collection of expressed proteins involved in the oxidation of reduced sulfur compounds that appear to originate from the recently described SUP05 clade of gamma-proteobacteria that is abundant in oxygen minimum zones. In contrast, communities in the deep waters of the stably hypoxic SLE were characterized by expression of proteins evidently originating from the uncultivated SAR324 clade. The SAR324 expressed proteome was comprised of numerous ABC-type transporters for organic compounds, including peptides and sugars, suggestive of an active heterotrophic metabolism. These findings are shedding light on the metabolic pathways active underin situ conditions and their regulation by oxygen and nutrient availability. Further time-series based studies will be used to describe the temporal variability of the microbial community proteome in the context of environmental forcing.

346A Biogeographic partitioning of Southern Ocean picoplankton David Wilkins*, Federico M Lauro, Timothy J Williams, Ricardo Cavicchioli University of New South Wales, Australia

The Polar Front, the major front of the Antarctic Circumpolar Current, forms an oceanographic boundary in the Southern Ocean between the cold Antarctic Zone and the warmer Subantarctic. There

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is growing evidence that the Circumpolar Current is warming, freshening and moving southwards due to climate change, but the effect this will have on the microbial ecosystems of the Southern Ocean is poorly understood. We performed a high-throughput metagenomic survey of Southern Ocean picoplankton during the austral summer of 2007-2008 and found that the Polar Front was a major biogeographical boundary, separating microbial assemblages with distinct taxonomic and functional profiles. Using a novel bioinformatic technique for reducing false positives when associating metagnomic reads with species, we observed a strong over-representation of the oligotrophic SAR11 and SAR116 clades, the cyanobacterial genera Prochlorococcus and Synechococcus, and Roseobacter spp. north of the Polar Front, and an overrepresentation of Bacteroidetes, the Gammaproteobacterial Sulfur Oxidizer-EOSA-1 clade and ammonia oxidising archaea characterised waters south of the Polar Front. In addition, we examined the partitioning of gene function by the Polar Front. Genes related to histidine degradation, biotin and cobalamin biosynthesis, proteasomes and lipopolysaccharide metabolism were overrepresented south of the Polar Front, while photosystem II and aminomethyltransferases linked to DMSP metabolism were overrepresented to the north. These results are consistent with the high nutrient, low chlorophyll status of the Subantarctic and dominance of eukaryotic phytoplankton and associated bacteria in the Antarctic Zone, and suggest differences in iron and nitrogen acquisition strategies.

347A Interactive climate change and runoff consequences alter O2 fluxes and bacterial community composition of coastal biofilms from the Great Barrier Reef Verena Witt*1, Christian Wild2, Sven Uthicke3 1Australian Institute of Marine Science (AIMS)/Leibniz Center of Tropical Ecology (ZMT), Germany, 2Leibniz Center of Tropical Ecology (ZMT), Germany, 3Australian Institute of Marine Science (AIMS), Australia

Global and local anthropogenic disturbances affect coral reefs worldwide. Coastal inshore regions of the Great Barrier Reef (GBR) receive substantial amounts of land runoff from local agriculture that lead to deterioration in water quality, specifically reduced light and increased nutrient availabilities. Land runoff may have interactive effects with ocean warming and could result in shifts of coral reef communities. Associated microbial biofilms are potential bioindicators for changes in water quality as they respond rapidly to environmental changes. We therefore investigated quantitative (C-, N- and chlorophyll a contents) and qualitative (microbial community composition) parameters in combination with metabolic responses (O2 fluxes) of biofilms established on glass slides. Biofilms were exposed to combinations of manipulated water temperatures (26, 29, 31°C), nitrate (0.5, 1.0, 1.4µM) and light availabilities (40, 200 μmol photons m-2 s-1) in a 28d flow-through aquarium experiment. These factors simulated nearshore conditions (low light/ high nitrate availability) found in situ during the dry season (26°C, 0.5µM) and during flood plumes in the wet season (29°C, 1.4µM), while the combination high light/ low nitrate availability represented situations further offshore. Findings revealed that, independent of light availability, 31°C significantly decreased net O2 production, C-, N- and chlorophyll a contents of biofilms. Under high light, additive effects of high temperature (31°C) and high nitrate (1.4µM)significantly reduced net O2 production. Terminal restriction fragment length polymorphisms (T-RFLP) of 16S rRNA genes showed microbial community shifts induced by high temperature that were driven by increases in the relative abundance of Oceanospirillum (Gammaproteobacteria) and decreases of Cyanobacteria. Only in high light-exposed biofilms, nitrate-induced community shifts were caused by increases in relative abundance of diatom plastids, while low light-exposed biofilm communities remained unaffected by elevated nitrate concentrations. Under high light, high temperatures had the most distinct effect on bacterial community composition, as elevated nitrate concentrations were only important in combination with 31°C. Further, high light-exposed biofilms became nitrate-limited as illustrated by significantly increasing net O2 production at 1.0μM nitrate. In contrast, low light-exposed biofilms were presumably light-limited; neither community composition nor productivity changed under increased nitrate availability. High temperatures altered microbial biofilm community composition, biomass and productivity and interacted with terrestrial runoff. Thus, under predicted near-future inshore reef scenarios (low light/ high nitrate availability), biofilms will become light-limited through sedimentation from runoff. However, at offshore reefs (high light/ low nitrate availability), biofilms will remain nitrate-limited, yet only under the assumption that climatic effects will not worsen weather events and flood plumes will not reach these reefs. The observed rapid community shifts in key microbial groupsmay contribute to bioindicator development for water quality in tropical coral reefs. Understanding the interactive effects of environmental changes on microbial biofilm communities may have broad implications for coral reef ecosystem functioning and development of improved coastal management strategies.

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348A Metagenomic insights on virioplankton biology through nucleic acid and protein synthesis genes K. Eric Wommack*1, Shawn Polson1, Shannon J. Williamson2, Eric Sakowski1, Helen Schmidt1, Mara Hyatt1, Jeff Wray1, Rachel Marine1 1University of Delaware, USA, 2Lake Pend Oreille Waterkeeper, USA

Ground-breaking viral ecology research has answered many primary questions surrounding the ecological role of aquatic viruses. For productive environments, we have a quantitative appreciation that high viral production rates sustain abundant virioplankton communities; and that viral infection and lysis significantly influence carbon and energy flux in aquatic microbial food webs. However, deeper questions of how the composition and diversity of virioplankton influences these emergent impacts of viral infection and lysis have been more intractable. Recently, shotgun metagenomic analyses have provided wholesale access to detailed genetic information and thus a means to addressing difficult second order questions. To date, shotgun metagenomics has been used to examine the diversity of virioplankton assemblages and ask how virioplankton diversity and composition changes across environments. However, metagenomic sequence data can also provide insights on the predominant biological features of viruses that have a direct influence on ecosystem-scale processes.

Through sequence clustering and supervised assembly we have conducted careful phylogenetic analysis of proteins involved in nucleic acid and protein synthesis. Unlike viral structural proteins which are exclusive to viruses, nucleic acid and protein synthesis genes are found throughout viral and cellular life and thus provide a link between viral and cellular evolutionary history. Moreover, these genes are under strong selective pressure as they play a critical role in viral replication. Phylogenetic analysis of full-length DNA polymerase I sequences revealed extraordinary diversity of this protein within the virioplankton, a diversity far greater than that known for bacteria. While homologs to familiar phage (for example coliphage T7) were found, the majority of DNA pol I sequences fell outside well known clades. Surprisingly, clades of virioplankton DNA pol corresponded well with predicted biochemical features of the enzyme and viral lifecycle. As a consequence, we predict that most marine phages carrying DNA pol I are lysogenic, not lytic, and thus have a less direct impact on C and nutrient cycling.

The frequency of different classes of ribonucleotide reductase (RNR), a protein which supplies reduced ribonucleotides for DNA replication, changed with environmental context and in correspondence with water column oxygenation. Oxygen-independent Class II RNRs were more common at a sub-tropical site and decreased in abundance moving towards higher latitudes. Oxygen-dependent Class I RNRs were similarly abundant at temperate latitude sites. Among known phages, chaperonins are relatively rare, however, homologs of these genes critical to protein folding appear to be common in the virioplankton. Many more viruses appear to carry the small subunit chaperonin GroES than the large subunit GroEL possibly indicating that viruses use this gene to alter the folding capabilities of the host GroEL. Assembly analysis indicated that no viruses carried both genes, a finding consistent with known viruses. These investigations would not have been possible without shotgun metagenomic sequence data as a posteriori, PCR-based approaches could not have captured the diversity of these protein coding genes. Together these findings show how detailed phylogenetic analysis can advance fundamental understanding of important biological features of viruses within microbial ecosystems.

349A Biodiversity and phylogenetic analysis of culturable bacteria of five brine pools of the Red Sea Guishan Zhang*, André Antunes, Yue Guan, Tyas Hikmawan, Uli Stingl 1Red Sea Research Center, King Abdullah University of Science and Technology (KAUST), Saudi Arabia

The Deep-Sea Brine Pools at the bottom of the Red Sea are some of the most remote and extreme environments on the planet. Elevated temperatures, high salinity, high concentrations of heavy metals and anoxia are some of the common characteristics of these habitats. We investigated the biodiversity of culturable bacteria of five brine pools (Nereus, Erba, Discovery Deep, Atlantis II, and Kebrit) using heterotrophic media (Marine broth and starch, casein), and were able to isolate 185 strains. An analysis of the 16S of the isolates showed that most of the isolates belonged to different species of 10 genera (Idiomarina, Marinobacter, Chromohalobacter, Halomonas, Halanaerobium Halobacillus,

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Zunongwangia, Halanaerocella, Alcanivorax, Rhodobacter), with five of them representing potential novel species specific to the brines. The isolates are being tested in an on-going anti-cancer screen, and will be tested for the production of enzymes degrading cellulose, petroleum, and aromatic compounds, all of which play an important role in ecological balance of the brines and are of high commercial interest. Based on this study it can be concluded that brine pools are populated with diverse culturable bacterial groups, and that the isolates are potential sources of extremely stable enzymes for commercial applications.

350A Response of bacterioplankton to glucose gradient without lysis and grazing: who is the winner in the competition for organic carbon? Rui Zhang*1, Markus Weinbauer2, Pei-Yuan Qian3 1Xiamen University, China, 2Université Pierre et Marie Curie-Paris 6, France, 3Hong Kong University of Science and Technology, China

Bacterial utilization of dissolved organic matter (DOM) plays an important role in marine carbon cycling. In this study, response of bacterioplankton to a gradient of carbon (glucose) addition was investigated in the maximum elimination of top-down control of viruses and flagellates in a subtropical coastal environment. Bacterial abundance was stimulated and corresponded to the gradient addition of glucose while the response of bacterial production was not significant, suggesting different bacterial life strategies under different nutrient conditions. Bacterial communities revealed by both terminal restriction fragment length polymorphism (TRFLP) and denaturing gradient gel electrophoresis (DGGE) showed a hump-like productivity-diversity relationship after three-day incubation. Both DNA fingerprinting profiling and cluster analysis showed clear and gradual changes in bacterial community structure along the gradient of glucose concentrations, reflecting the competition for carbon supply among bacterial groups. Sequencing analysis of DGGE bands disclosed the relative abundance of seven bacterial genotypes in Alteromonadaceae and Roseovarius that gradually decreased with the glucose enrichment while two Vibrio genotypes showed the reverse increasing trend. This suggested that Vibrio was a more successful opportunist at high carbon availability. Our study provided novel information of marine bacterial utilization of DOM by using virus reduction bioassay.

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