tuesday 21 august ps14 – microbial community diversity ...llebbe/abstracts/isme 14 abstract...

Tuesday 21 August

1 PS14 – Microbial Community Diversity

PS14 – Microbial Community Diversity: 16S and Beyond - Main Authors D – M

248B Analyzing microbial diversity using the metagenomics tools of BioNumerics® Katrien De Bruyne, Johan Goris, Katleen Vranckx*, Hannes Pousseele, Koen Janssens Applied Maths NV, Belgium

The advent of low cost next-generation sequencing methods is having a profound effect on the study of the microbial world, and not in the least on the field of metagenomics and the study of uncultured bacterial diversity. Direct investigation of the vast majority of bacteria, irrespective of their culturability and taxonomic identities, has challenged the classical concept of species diversity and has led to various new insights into the composition and functionalities of microbial communities. Numerous studies in the field of microbial ecology, biodiversity, bioremediation, bioprospection and medicine have already been published. To analyze and assess the microbial diversity, most of these studies use open source packages such as mothur, pyronoise/ampliconnoise, vitcomic and qiime or use a variety of (in-house developed) scripts.

We recently developed a tool in the BioNumerics® software suite for the analysis, quantification, visualization and comparison of microbial communities starting from sequence reads, independent of the sequencing platform used. Within the graphical user interface, the concept of a metagenomics analysis workflow is used to which actions can be added or deleted to their own interest, e.g. filtering of the reads, calculating operational taxonomic units, computing sample statistics, calculating diversity indices, …

The BioNumerics® program makes use of the mothur project, initiated by Dr. Patrick Schloss and colleagues (Department of Microbiology & Immunology, The University of Michigan). The mothur project filled in the needs of the microbial ecology community by incorporating the functionality of numerous application e.g. dotur, treeclimber, s-libshuff, Unifrac into one command line application. BioNumerics® uses the flexibility of the algorithms implemented in mothur and further elaborates on these results by creating a fully interactive reporting service including a geographical visualization tool and various chart tools for the interpretation and manipulation of the results. The integrated follow-up analysis includes an environment for elaborated data mining and statistics. In this study, we illustrate this new tool with publicly available genomic data sets.

In conclusion, the BioNumerics® software offers one environment to start from the raw read sequences; perform trimming, chimera removal and sequence clustering to end up with visualization of the operational taxonomic unit abundances or the evaluation of the α- and β-diversity using a plethora of indices. Performing each analysis step is facilitated by an intuitive user interface and visual feedback on the analysis results using flexible reporting tools. The integration of the metagenomics functionality in the existing BioNumerics® software package enables the combination of the formerly used environmental analyses such as DGGE, TGGE, or ARISA with the newly obtained metagenomics results to allow comparison between the different methods. This stresses the importance of BioNumerics®, as a universal platform for databasing and integrated analysis, including a wide range of data mining, clustering, identification and statistical tools adding much more flexibility, traceability and support in comparison with a series of separate (script-based) analysis tools.

249B Metagenomics for practical management of microbial drinking water quality Aleida de Vos van Steenwijk*, Janneke Krooneman, Bert Geurkink, Gijs Faber, Inez Dinkla Bioclear, Netherlands

Drinking water companies are responsible for the supply and quality of our drinking water. One of their main ambitions is therefore is to keep the quality of the drinking water stable from water treatment facility to customer. This is feasible for chemical and physical properties, but proves to be more difficult for microbial properties. These can vary throughout transport through the drinking water distribution network mainly through unwanted (re)growth of micro-organisms. Drinking water companies increasingly want insight into aspects such as (i) Why do microbial parameters vary? (ii) How can microbial (re)growth be minimised? (iii) Which parameters should be measured to monitor microbial risks? In this presentation a case will be presented in which next generation sequencing (Illumina) and a newly developed bioinformatics tool called BioProphyler® were used to gain detailed insight in the

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microbial diversity in the distribution network. The presentation will show how this approach forms a vital stepping stone for practical solutions in management of the microbial quality of our drinking water.

In order to achieve the ambitions of drinking water companies a detailed insight is needed in the diversity and composition of organisms in the drinking water distribution network. Methods most frequently used outside the research world are not capable of achieving the required level of detail. Metagenomics tools are more than capable of achieving this, but are almost exclusively used in fundamental research and application. This is owing to the fact that the metagenomics techniques are complex and that the amount of data generated is very large. Furthermore, in most cases it takes too long to gain practical answers and solutions to questions in the ‘real world’ and costs associated with such an approach are high. The gap that needs to be bridged between science and business is very large.

Currently, drinking water companies in the Netherlands are required, by law, to monitor the quality of drinking water using culturing techniques for species of Aeromonas and other indicator organisms. However this approach cannot provide a total picture of the microbial situation in the distribution network. Furthermore quantitative molecular analyses (Q-PCR) indicated that - in eleven samples analysed - a maximum of 0.6% of the bacterial population consisted of species of Aeromonas. This led to the question what the other 99.4% of the population consists of...

Firstly, to answer this question, an approach using Denaturing Gradient Gel Electrophoresis (DGGE) and DNA sequencing was used. With this approach only 13 species in the complex ecosystem were actually identified (5 bacteria and 8 Archaea). It became clear that this approach is too time-consuming and cannot provide sufficient detail in this instance.

Consequently an approach using next generation sequencing (Illumina) and a new bio-informatics tool called BioProphyler® (developed by Bioclear) were used. Using this novel and unique approach a detailed insight into the diversity of organisms in drinking water and sediment at a location in the drinking water distribution network was obtained.

250B Microbiome of Brazilian mangrove sediments as revealed by metagenomics Armando Dias*1, Diego Javier Jiménez2, Diego Chaves2, Danice Mazzer Luvizotto3, Francisco Dini-Andreote3, Cristiane Cipola Fasanella3, Maryeimy Varon Lopez3, Sandra Baena4, Rodrigo Gouvêa Taketani5, Itamar Soares Melo5, Fernando Dini Andreote3 1University São Paulo - USP, Brazil, 2Colombian Corporation of Agricultural Research (CORPOICA), Colombia, 3Department of Soil Science, “Luiz de Queiroz” College of Agriculture, University of São Paulo, Brazil, 4Colombian Center for Genomic and Bioinformatics from Extreme Environments (GeBiX), Colombia, 5Laboratory of Environmental Microbiology, EMBRAPA Environment, Brazil, Brazil

Here we embark in a deep metagenomic survey that revealed the taxonomic and functional aspects of mangrove sediment microbiology. The extraction of DNA from sediment samples and the direct application of pyrosequencing resulted in approximately 215 Mb of data from four distinct mangrove areas (BrMgv01 to 04) in Brazil. The taxonomic approaches applied revealed the dominance of Deltaproteobacteria and Gammaproteobacteria in the samples. Paired statistical analysis showed higher proportions of specific taxonomic groups in each dataset. The metabolic reconstruction indicated the possible occurrence of processes modulated by the prevailing conditions found in mangrove sediments. In terms of carbon cycling, the sequences indicated the prevalence of genes involved in the metabolism of methane, formaldehyde, and carbon dioxide. With respect to the nitrogen cycle, evidence for sequences associated with dissimilatory reduction of nitrate, nitrogen immobilization, and denitrification was detected. Sequences related to the production of adenylsulfate, sulfite, and H2S were relevant to the sulphur cycle. These data indicate that the microbial core involved in methane, nitrogen, and sulphur metabolism consists mainly of Burkholderiaceae, Planctomycetaceae, Rhodobacteraceae, and Desulfobacteraceae. Comparison of our data to datasets from soil and sea samples resulted in the allotment of the mangrove sediments between those samples. The results of this study add valuable data about the composition of microbial communities in mangroves and also shed light on possible transformations promoted by microbial organisms in mangrove sediments.

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251B Consequences of agricultural land use in semi-arid ecosystems for soil bacterial diversity revealed by genetic profiling of 16S rRNA genes Guo-chun Ding*1, Yvette Piceno2, Nicole Weinert1, Holger Heuer1, Anja Dohrmann3, Angel Carrillo4, Thema Castellanos4, Christoph Tebbe3, Kornelia Smalla1 1Julius Kühn-Institut, Germany, 2Earth Sciences Division, USA, 3Biodiversity, Johann Heinrich von Thünen Institute, Germany, 4Centro de Investigaciones biologicas del Noroeste, Mexico

In semi-arid ecosystems, transition from natural land to agricultural use resulted in the loss of characteristic flora and fauna. In the present study, the long-term (ca. 50 years) effects of such transition on soil bacterial community were explored at two sites typical for semi-arid desert. Comparisons were made between samples from alfalfa fields and the adjacent natural scrublands. Amplified 16S rRNA gene fragments from total community DNA were analyzed by a suite of complementary approaches. Denaturing gradient gel electrophoresis analysis revealed significant effects of the transition on community composition of Bacteria, Alpha-, Betaproteobacteria, and Actinobacteria at both sites. PhyloChip analysis uncovered that the transition affected negatively oligotrophic or anaerobic taxa such as Acidobaceria, Chloroflexi, Acidimicrobiales, Rubrobacterales, Deltaproteobacteria and Clostridia, whereas it had a positive effect on copiotrophic groups (Alpha-, Beta-, Gammaproteobacteria, Bacteroidetes and Actinobacteria). Compared with the natural soil from scrubland, the agricultural soil was lower in organic matter and phosphate concentration but higher in salinity. Redundancy analysis suggested that the community composition of taxa responding to the transition (except for Spirochaetes) was highly related to soil parameters which were significantly different between the agricultural soil and the natural soil. Overall, this study provides insights into the influence of land use on soil bacterial community.

252B Restoration process of a coal mining soil influences microbial community diversity, abundance, and composition Patricia Dorr de Quadros*1, Kateryna Zhalnina2, Austin Davis-Richardson2, Jennie Fagen2, Flavio Camargo1, Eric Triplett2 1Federal University of Rio Grande do Sul, Brazil, 2University of Florida, United States

Candiota, the biggest coal mine of Latin America, is located in South Brazil and generates about 350 megawatts of energy producing about 1.7 million tons of coal a year. Coal mining makes an important contribution to the social and economic development of the region; however, mining disturbs large areas of land and raises a number of environmental concerns. The process of coal extraction usually results in contamination of soil layers with pyrite, which is oxidized forming acidic compounds. The resultant low pH causes the dissolution of minerals, increasing the concentration of metals like Al, Fe, Mn, Cu, Ni and Zn to toxic levels. Additional environmental disturbances associated with the coal mining process include: soil compaction, erosion, dust, noise, water pollution, and impacts on local biodiversity. The goals of this work were to evaluate the changes on soil microbial diversity, abundance, and composition in the Candiota coal mining soil restoration area. Sites at different stages of restoration (3, 4, 5, 6 and 19 years after coal extraction) were compared with undisturbed soils (forest and native field) and correlations with soil features including chemical compounds, soil heavy metals content, microbial biomass and enzyme concentrations of the soils were examined. Within bacteria and archaeal domains, 24 phyla, 46 classes, 95 orders, 191 families, 525 genera and 2005 species were detected. At phylum level, 7-fold less OTUs were detected in reconstructed soils than in forest and natural pasture soils, exhibiting a drastic decrease of microbial diversity after soil reconstruction. The presence of Bacillus, Sphingomonas, Streptomyces and Desulforomonas were the most abundant at reconstructed sites. Actinobacteria were positively correlated with copper concentration on soils. It was found that soil from recent reconstructed areas have lower microbial diversity, microbial biomass, and desidrogenase and beta-glucosidase concentrations compared to non-disturbed areas. The differences of microbial diversity and composition from 3 to 19 year old sites decreases over time, but does not reach the original levels observed at the undisturbed sites. This indicates that the present soil restoration processes cannot recover soil quality to that of undisturbed soil. The disorder caused by mining (increased temperature, moisture loss, loss of physical structure and organic matter) alters the soil microbial community. The characterization of the microbial population is sensitive enough to differentiate soils in regions mined for coal and can assist in determining rehabilitation methods for reconstructed soils.

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253B Pyrosequencing analysis of bacterial community structure in an experimental drinking water distribution system Isabel Douterelo*, Rebecca Sharpe, Katherine Fish, Joby Boxall The University of Sheffield, United Kingdom

Microbial biofilms formed on inner-pipe surfaces in drinking water distribution systems (DWDS) may affect the quality and safety of drinking water, particularly if mobilised from the pipe wall to the bulk water. The objectives of this study were to improve knowledge on bacterial ecology of DWDS by pyrosequencing analysis and to assess the potential mobilisation of biological material from pipe walls.

Experimental work was carried out in a full scale temperature controlled test-loop facility capable of reproducing the dynamics of real networks.The facility is fed from the local distribution system with a 24 hour water retention time to maintain a chlorine residual and nutrient supply. The facility made of high density polyethylene (HDPE) pipes, typical of UK distribution systems, run as three 200m long recirculating loops with individual control. HDPE coupons, designed to maintain the boundary layer hydraulics, were inserted around the mid-length of each loop. After 28 days of biofilm growth at steady state conditions and 16°C, the system was flushed by sequentially increasing the boundary hydraulic forces. Coupons and bulk water samples were collected pre‐ and post‐flushing for physico-chemical and DNA-based analysis. DNA was extracted from biofilm and water samples and bacterial tag-encoded FLX 454 pyrosequencing (bTEFAP) was performed by the Research and Testing Laboratory (Lubbock, TX, USA) using primers Gray28F and Gray519r which span the variable regions V1-V3 in the 16s rRNA gene.

Significant differences (p<0.05) in the relative abundance of bacteria at different taxonomic levels were detected between biofilm and water samples.Gammaproteobacteria (27-50%) at class level and Pseudomonas (up to 48%) at genus level predominated in biofilms, while Alphaproteobacteria (68-77%) and species belonging to the genera Methylocystis (23-31%) and Methylocella (17-21%) were abundant in water samples. Higher species richness (Chao estimator) and diversity (Shannon'sindex) were detected in pre-flushing biofilm samples than in water samples. The increase in water turbidity after flushing indicated that pipe-wall material was mobilised into the bulk water. However, no significant differences in the bacterial community composition were found between pre- and post-flushing water samples. Flushing did not completely removed biofilms from the pipes but altered the pipe‐wall bacterial community structure. After flushing, considerable shifts in Gammaproteobacteria (increased 23%) and Alphaproteobacteria (decreased 21%) abundance were observed. At genus level, the main changes in abundance were detected in Pseudomonas which decreased 25% and in Erythromicrobium (increased 13%). While flushing did not noticeably influence taxonomic richness, a decrease in species diversity was detected in post-flushing biofilm samples.

The use of pyrosequencing allowed for circumventing the limitations of methods currently used to assess the microbial quality of drinking water and revealed previously unexplored aspects of the bacterial ecology of DWDS, including the mobilisation of biofilms from pipe walls following changes in system hydraulics

254B Microbial methane oxidation in livestock slurry surface crusts: community structure and inhibition by N species Yunfeng Duan*1, Nico Boon2, Lars Elsgaard1, Søren O. Petersen1 1Aarhus University, Denmark, 2Ghent University, Belgium

Livestock slurry is an important source of agricultural methane emissions. During slurry storage, a floating surface crust may form naturally, or crust formation may be facilitated by the addition of straw. In this environment, methane-oxidizing bacteria (MOB) and a potential for methane oxidation have been found, suggesting that surface crusts could reduce methane emissions from slurry storages. However, little is known about the microbial ecology of MOB in surface crusts, or how they interact with environmental factors such as inorganic nitrogen.

The microbial diversity of three different slurry surface crusts (swine slurry natural crust, swine slurry straw-reinforced crust, cattle slurry straw-reinforced crust) was analysed using Denaturing Gel Gradient Electrophoresis. Band pattern analysis showed distinct structures of general bacterial, Type I & Type II MOB, and archaeal communities in all studied crust materials. The two swine slurry crusts

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harboured similar predominant species, which are often not found in cattle slurry crusts, indicating that slurry type had great impact on microbial community development. Straw addition enhanced microbial diversity, especially the richness of less abundant species. Type I MOB were much more diverse than Type II MOB in all studied crusts, suggesting that Type I MOB could be primarily responsible for methane oxidation in these crusts, in accordance with the expected high availability of methane. During spring 2012, pyrosequencing followed by bioinformatic analysis will be carried out to further investigate the identities and phylogeny of microorganisms in surface crusts, and 13C labeling of membrane lipids will be attempted to identify active MOB groups.

Inhibitory effects of ammonium, nitrate and nitrite on potential methane oxidation were investigated using crust material from a cattle slurry store. Methane oxidation rates were assayed at N salt concentrations up to 500 mM, and at headspace methane concentrations of 100 ppmv or 10,000 ppmv. First-order rate constants were calculated as indicators of the strength of inhibition, and the rate constants were fitted to a logistic dose-response model to reveal patterns of inhibition. The results showed that N species had different effects on microbial methane oxidation. Nitrite was the most potent inhibitor, reducing potential methanotrophic activity by up to 70% at just 1 mM. MOB were least sensitive to nitrate, and up to 50 mM nitrate was tolerated without any decline in methanotrophic activity. The inhibition by ammonium increased progressively with ammonium concentration, but it appeared to be a comparatively weaker inhibitor than nitrate at high concentrations, possibly due to sorption to the crust matrix. The higher methane concentration (that is 10,000 ppmv) significantly increased specific methane uptake rates, but did not change the inhibition patterns of N salts.

The kinetic parameters obtained in this and other studies suggest that methane oxidation in surface crusts are performed by high-concentration MOB – possibly Type I, which is also supported by our DNA fingerprinting analysis.

255B Deep into the bacterial communities living in soils cultivated with sugarcane in Brazil Ademir Durrer*1, Thiago Gumiere1, Mayra C A Costa1, Rodrigo G Taketani2, Fernando D Andreote1 1University of São Paulo, ESALQ, Brazil, 2Embrapa Environment, Brazil

The sugarcane is nowadays the most important crop in Brazil, where it is used as the basic material for the production of ethanol, sugar, plastic, and many other sub-products. It is also actual a demand to increase production, by either improving productivity or increasing the cultivation area. Considering this scenario, the search for sustainable alternatives is desirable, and one of the pillars in this context would it be to better explore the role of the microbial communities to support a better plant development, supplying it with nutrients, or inhibiting the occurrence of diseases and pests. However, little is known about the microbial community inhabiting the soils cultivated with sugarcane. In this way, this work aim to deeply describe the bacterial diversity present in soils cultivated with sugarcane in the State of São Paulo (Brazil) by a biogeography approach. Such attempt is based on a total of 114 samples, collected in areas with distinct plantations managements, distributed along 12 different producer regions. Data was generated by highthrouput sequencing of V6 region of the 16S rDNA gene using the IonTorrent platform. Using RDP Naïve Bayesian rRNA Classifier, with 50% of confidence threshold, the tags exhibited an average size of 98 pb, and the total of 3.260.815 sequences were obtained, along with 1.802.188 were classified into known bacterial phyla and 1.458.627 were just classified as non-classified Bacteria. Concerning the phyla-allocated sequences the results demonstrated that the most abundant phyla were: Proteobacteria, Actinobacteria and Acidobacteria, followed by Verrumicrobia and Firmicutes. The occurrence of these prevalent phyla was distinct among samples, what served as a source for clustering analysis which revealed the formation of seven distinct groups of soils samples. These results suggest a biogeographic distribution of bacteria along the analyzed samples, which is driven by geographic regions, and also by the physico- chemical characteristics of soils and managements adopted in the 12 analyzed regions. As a conclusion, the results demonstrated a highly diverse and responsive bacterial community living in soils cultivated with sugarcane, and does indicate this issue as a promising point to contribute on the increase of sugarcane productivity and sustainable cultivation.

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256B NCMA: A bioresource center for marine microorganisms David Emerson*, Willie Wilson, Michael Preston Bigelow Laboratory for Ocean Sciences, United States

The NCMA currently houses the world's largest marine phytoplankton collection with over 2500 strains of bacterial and eukaryotic phytoplankton. To more fully serve the entire community of marine researchers the NCMA has recently started to acquire bacterial and archaeal strains, as well as viruses that infect either microscopic eukaryotes or bacteria. For bacteria and archaea, the acquisition strategy is aimed at either specific groups of microbes, e.g. hydrocarbon degraders or fish pathogens, habitats, e.g. the open ocean, or processes, e.g. nitrification or sulfate reduction. New strains are accessioned into both seed stocks and distribution stocks, purity is confirmed, and identity authenticated by microscopy, physiology, and molecular analysis. A website (ncma.bigelow.org) provides an easy portal for depositing strains, searching the collection, and ordering materials. The website also provides extensive meta-data for strains including geographical and physiological data. In addition to offering the traditional services of a culture collection; the NCMA has a goal of providing standards that will aid researchers in the next generation of microbial community analysis. These include 'mock communities' for analysis of pyrosequencing data where known 16S or 18S amplicon concentrations of community members are provided to run as standards; 2) amplicons from specific microbe(s) for quantitative PCR (qPCR) analysis; 3) controls to help evaluate cell lysis efficiency and/or yield recovery. We are in the process of validating such controls from open ocean and benthic marine communities. This work will provide resources for the expanding revolution in microbial ecology, and allow researchers to better quantitate their results when analyzing marine microbial communities.

257B Effects of extremely short sludge retention time and short hydraulic retention time on microbial community in an external membrane bioreactor Ipek Ergal*, Mert Kumru, Halil Kurt, Senem Teksoy Başaran, Seval Sözen, Derin Orhon, Alper Tunga Akarsubaşı Istanbul Technical University, Turkey

The major issue in operation of biological wastewater treatment systems in terms of effluent standards and volume requirements is the high biomass concentration in the system. Membrane Bioreactors (MBR) have been preferred for their ability to hold high biomass concentrations by employing long sludge retention time (SRTs) in the systems without facing any limitations due to biomass control and/or settling problems.

It has been suggested that the increases in SRT could promote the development of slow growing microorganisms since they are able to consume the macro-molecules (polysaccharides, carbohydrates, and protein) as substrates and produce less biopolymers.

The concept of biological wastewater treatment systems with low biomass concentration (i.e. short SRT) promises a totally recoverable treatment scheme conserving energy and meeting the discharge criteria. However, there are few studies to elucidate the effect of very short SRT on the microbial composition of the MBRs.

The aim of experimental work presented in this study is, to evaluate the carbon removal performance of the 'high rate external-MBR (EMBR)' operated at extremely short sludge retention time (SRT) (0.5-2 d) and hydraulic retention time (HRT) (0.5-2 hr) and also to associate the microbial community change with the performance of the reactors.

For this purpose, the EMBRs were run in successive experiments at SRT of 0.5, 1 and 2 days where reactors were operated at three different HRTs (0.5-1-2 hr) for each SRT.

Synthetic substrate solution, representing the readily biodegradable chemical oxygen demand (COD) fraction of domestic sewage, which has a total of 200 mg COD/L is prepared as wastewater feed in the experiment. The COD of the samples were analyzed according to ISO6060 methodology.

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Samples were collected from each successive reactor operation at the start up and final stages which was considered as steady state (over 90% COD removal). Population changes were monitored by 16S rDNA gene based PCR-DGGE methodology. DGGE patterns were analyzed with BioNumerics software 5.1. Similarity matrix and dendogram of the DGGE profiles were generated based on the Dice correlation coefficient (band-based) and unweighted pair-group method with arithmetic mean (UPGMA).

The structural diversity of the microbial community was examined by the Shannon index of general diversity H.

The mean of similarity between start up and final samples for SRT=0.5d was 81.39%±5.1 and 72.52%±5.6 for SRT=1d and 77.48%±0.8 for SRT=2d, p=0.02. According to pairwise similarities, microbial profiles between start up and final samples shifted least for SRT=0.5d compared to 1d and 2d.

Microbial diversity of the final samples for SRT=0.5 days 1.12±0.09 was higher than the mean value of the diversity index for the reactor samples with SRT=1 and 2 days 1.07±0.03. Corresponding relationship is also observed for number of detected DGGE bands for which the values were 17.7±2 and 15.6±1 respectively, p=0.15.

Regardless of the fact that SRTs were short, there were significant differences of bacterial diversity especially 0.5 days versus 1 and 2days. In spite of variation in the microbial community, COD removal efficiencies have not been changed significantly.

258B Molecular characterization of partial nitrification followed by anammox process Elif Esen*1, Duygu Dityapak2, Halil Kurt1, Mert Kumru1, Bilge Alpaslan Kocamemi2, Neslihan Semerci2, Alper Tunga Akarsubaşı1 1Istanbul Technical University, Turkey, 2Marmara University, Turkey

The Anammox process is an anaerobic chemolithoautotrophic biological conversion process mediated by a group of Planctomycetes bacteria associated with at least five genera. The process converts ammonium directly into dinitrogen gas under anaerobic conditions using nitrite as the electron acceptor in the absence of any organic carbon source. In the past decade, the Anammox process has been applied widely in lab scale studies for the treatment of ammonium-rich and low organic carbon containing wastewaters, e.g., sludge digester supernatant. However there are few studies about the Anammox process combined with partial nitrification process.

In this study, to obtain the desired NO2-N:NH4-N influent ratio of Anammox reactor, partial nitrification process is implemented before Anammox process (PN-SBR system). Also the association between the microbial community changes and the performance of the reactors was investigated in parallel with operation of reactors. Eight samples from Anammox system and 10 samples from partial nitrification system were collected in each steady state. 16S rRNA gene based PCR-DGGE methodology was used to observe the population changes in the reactors. Both partial nitrification and Anammox reactor, the eubacterial community similarities between first and last samples are 85.43% and 31.79%, respectively. Fluorescence in-situ hybridization (FISH) method has been used to detect ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) in partial nitrification system. Nitrospira-like nitrite oxidizing bacteria species and Betaproteobacterial ammonia-oxidizing bacteria species has been detected among the abundant species in partial nitrification system. On the other hand Candidatus Scalidua wagneri, Candidatus Scalidua sorokinii, Candidatus Brocadia anammoxidans, Candidatus Kuenenia stuttgarteiensis species are determined in Anammox reactor.

The soluble biodegradable portion of COD is removed about 76% in all operating conditions. This indicates that almost all of the readily biodegradable portion of COD is removed in PN-SBR. So, the influent COD to the Anammox-SBR system mainly consisted of nonbiodegradable and/or slowly biodegradable soluble COD can not result in activation of denitrifiers significantly in Anammox-SBR system. In this study, the enrichment of Anammox bacteria from activated sludge of a local sewage treatment plant was successfully conducted as proven by molecular methods.

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259B Culture independent analysis of the colonic bacteriota during a long term probiotic feeding study James Evans*1, Julian Marchesi1, Eshwar Mahenthiralingam1, Sue Plummer2, Iveta Garaiova2 1Cardiff University, United Kingdom, 2Cultech, United Kingdom

It is becoming increasingly evident that the gut bacteriota extend an influence on the host far beyond their niche; with the potential to affect host health. It therefore follows that modulation of the host bacteriota could promote an individual's health. Modulation of the host bacteriota in many studies has been achieved through dietary supplementation with probiotics, but for only short periods, e.g. weeks. Here we report the development of culture independent methods for detecting changes in colonic bacteriota during a 6 month probiotic feeding study.

A cohort of 30 males were fed a probiotic mixture (2 strains of Lactobacillus acidophilus, Bifidobacterium lactis and B. bifidum at 109 cfu per capsule) for 6 months and stool samples taken before, during and after feeding. DNA was extracted from all samples and subjected to LH-PCR and ARISA to generate individual bacteriota fingerprints.

Both LH-PCR and ARISA were reproducible, distinguishing individuals and showed changes in colonic bacteriota fingerprint profiles over time.

Both LH-PCR and ARISA show promise as tools to analyse the colonic bacteriota of individuals. These methods will be applied to answer whether the long-term consumption of a probiotic supplement can impact upon the gut bacteriota or not.

260B Stable isotope probing and metagenomics of active DMS degraders from terrestrial environments Ozge Eyice*, Moto Namura, Yin Chen, Hendrik Schäfer University of Warwick, United Kingdom

Methylated sulfur compounds including dimethylsulfide (DMS), dimethylsulfoxide (DMSO) and methanethiol (MT) play a crucial role in the global sulfur cycle. With major emissions of DMS to the atmosphere, this volatile compound affects atmospheric chemistry and contributes to the atmospheric aerosol burden, which plays a role in climate-cooling.

Degradation by microorganisms is thought to be the main sink of DMS in terrestrial habitats. Previous studies have identified several bacteria from terrestrial environments such as Thiobacillus and Hyphomicrobium spp. but also methanogens which can use DMS as carbon source. However, the in situ diversity of DMS-degrading microorganisms is poorly characterized due to the lack of cultivation-independent studies.

In this study we sought to identify the aerobic bacterial populations actively degrading DMSas the carbon and energy source in a soil and a lake sediment using stable-isotope probing (SIP) and metagenomics. Soil and sediment sampleswere incubated with [13C2]-DMS or [12C2]-DMS and the labelled (heavy) DNA representing DMS-assimilating organisms was separated from the rest of the community (light) DNA by ultracentrifugation.

SIP incubations were terminated after consumption of different amount of [13C2]-DMS (approximately 5, 15 and 30 µmoles of carbon for soil; 10, 20 and 40 µmoles of carbon for lake sediment) in a time-course experiment. In order to evaluate the community profiles of ‘heavy’ and ‘light’ DNA fractions, denaturing gradient gel electrophoresis (DGGE) fingerprinting was applied. Sequencing of dominant DGGE bands indicated that Methylophilaceae family members and Thiobacillus thioparus were dominant taxa in 13C-DNA fractions in both the soil and lake sediment microcosms. Thiobacillus thioparus is a known DMS-degrader, however members of Methylophilaceae are known mainly as methanol and methylamine degraders and were not recorded as DMS-degraders previously.

A metagenomic analysis of the DNA from the SIP experiment was then carried out. Multiple displacement amplification was applied to obtain sufficient DNA for metagenomic analysis by high-throughput sequencing on the Illumina platform. Preliminary analysis of metagenomic sequence data

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showed the dominance of Thiobacillus and Methylophilaceae amongst the dominant taxa, confirming data obtained by DGGE. Additional metagenomic sequencing is currently being carried out to obtain insight into functional aspects of these potential DMS-degrading Thiobacillus and Methylophilaceae populations.

Overall the results of our study suggest that Thiobacillus and Methylophilaceae strains may contribute to DMS cycling terrestrial environments. While Thiobacillus spp. are amongst confirmed DMS-degraders, members of the Methylophilaceae family are methylotrophs and have significant metabolic potential, but no DMS-degrading isolates have been described so far. The SIP data suggest that they also potentially contribute to DMS degradation in terrestrial habitats. Alternatively, Methylophilaceae detected here may use intermediate compounds (such as MT, formate, formaldehyde) released by other DMS degraders in the sample, such as Thiobacillus spp.

261B Are microbial communities of subsoils influenced by agricultural management? Doreen Fischer*1, Timo Kautz2, Ulrich Köpke2, Jean Charles Munch3, Michael Schloter4 1Helmholtz Zentrum Muenchen , Germany, 2University of Bonn/ Institute of Organic Agriculture, Germany, 3Technical University Munich/Chair for Soil Ecology, Germany, 4Helmholtz Zentrum Muenchen/Environmental Genomics, Germany

Whereas it is well accepted that microbial diversity and activity in topsoils are highly influenced by agricultural management, little is known about how different cropping sequences or different fertilizer amounts and quality affect microbial life in subsoil systems, and in turn if this influences plant nutrition and performance in the long run mainly in organic farming systems. Therefore, it was the aim of this project to investigate microbial diversity in subsoils of different cropping sequences using molecular tools like community fingerprinting and high-throughput sequencing. However, our first results show that the structure of bacterial communities obtained via tRFLP fingerprinting depend on soil depth only, not on previous crops. Significant differences between depths of 30 cm (topsoil), 60 and 90 cm (subsoil) were observed.

262B Short-term temporal changes in community structure overwhelm changes from phosphorus addition in bacterioplankton communities of the Eastern Mediterranean Stelios Fodelianakis*1, Tron Frede Thingstad2, Panagiotis Kasapidis3, Paraskeyi Pitta3, Ioannis Karakassis1, Emmanuel Ladoukakis1 1Department of Biology, University of Crete, Greece, 2University of Bergen, Department of biology, Norway, 3Hellenic Centre for Marine Research, Greece

Eastern Mediterranean Sea is extremely oligotrophic and has profound phosphorus limitation for bacterial production and growth. A previous large-scale experiment in this area revealed that phytoplankton community was nitrogen and phosphorus co-limited while the heterotrophic community was phosphorus limited. A fundamental, yet unanswered question regards the factors that shape microbial communities in such oligotrophic environments. Here, we set up a mesocosm experiment to study short-term effects of phosphorus addition in bacterioplankton communities in this P-starved system in which we added inorganic phosphorus to 10m-deep pelagic water from the North coast of Crete island, Greece. We analyzed a total of 12 samples (three P-amended and three control mesocosms in experimental days 0 and 3) using 16SrRNA amplicon pyrosequencing of the V3 hypervariable region. We found that phosphorus addition has a minimal effect on community structure, limited only to subtle changes in certain OTUs’ relative abundances while not affecting species richness, evenness or the relative abundances of the most abundant groups in low phylogenetic resolution. On the contrary, time spent within the water tanks was found to have significant effects on community structure, altering all the above mentioned community traits. We therefore conclude that in short term, temporal changes in community structure overwhelm changes from phosphorus addition in this phosphorus-starved ecosystem.

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263B Metagenomic analysis of water supply in geographically defined areas of low and high incidence rates of inflammatory bowel disease in Manitoba, Canada Jessica Forbes*1, Gary Van Domselaar2, Charles Bernstein3, Denis Krause4, Ehsan Khafipour4 1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Canada 2National Microbiology Laboratory, Public Health Agency of Canada, Canada, 3Department of Internal Medicine, University of Manitoba, Canada, 4Department of Animal Science, University of Manitoba, Canada

Inflammatory bowel disease (IBD) represents a group of chronic idiopathic intestinal diseases characterized by dysbiosis of the gut microbiome. IBD is predominant in the northern hemisphere, with high incidence rates reported in Manitoba, Canada. Geographical areas with low and high incidence rates of IBD (LIA and HIA, respectively) have previously been mapped out across Manitoba. As such, we investigated whether the microbial composition of water from LIA and HIA are different and hence may represent a potential etiologic source. Water samples were collected from 20 LIA and 20 HIA in Manitoba. DNA was extracted and V1-V3 regions of bacterial 16S rRNA genes were amplified and sequenced using Roche 454 pyrosequencing. Analysis of pyrosequencing data was performed with the mothur software package. Sequences were converted to categorical data using a standard taxonomy by comparison to the Silva 16S rRNA database. Data were analyzed using generalized linear mixed-model methodology (GLIMMIX) of SAS for categorical data. Partial least square discriminant analysis (PLS-DA) was performed on genus data to identify genera that were most characteristic of LIA and HIA. A total of 10 phyla were represented in the dataset with Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes and Acidobacteria comprising approximately 98% of the total microbial composition. LIA was associated with higher populations of Proteobacteria, but lower populations of Firmicutes compared to HIA. Pseudomonas (23.0% of total), Sphingomonas (4.1% of total), and Herminiimonas (4.0% of total) represented the most dominant genera of the pooled metagenome. Among the bacterial taxa included in the model, Flavobacterium spp., Janthinobacterium spp., Propionibacteriaceae and Pseudomonadaceae were significantly associated with LIA while Mycobacterium spp., Bosea spp., Bradyrhizobium spp. and Actinomycetales were significantly associated with HIA. Several bacterial genera were associated with LIA or HIA. Future studies should investigate their role in pathogenesis.

264B Gut microbiome analysis of infected calves with low or high doses of Mycobacterium avium subspecies paratuberculosis (MAP) at different ages identifies specific bacteria associated with the outcome Jessica Forbes1, Jeroen De Buck2, Rienske Mortier2, Herman Barkema2, Steve Hendrick3, Gary Van Domselaar4, Denis Krause5, Ehsan Khafipour*5 1Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Canada, 2Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Canada, 3Western College of Veterinary Medicine, University of Saskatchewan, Canada, 4National Microbiology Laboratory, Public Health Agency of Canada, Canada, 5Department of Animal Science, University of Manitoba, Canada

Mycobacterium avium subspecies paratuberculosis (MAP) is the etiological agent of Johne’s disease (JD), a chronic enteritis of ruminants. While the etiology of JD is well established, it is unknown whether early changes in the gut due to infection have an impact on other gut microorganisms. As such, we characterized the gut microbiome composition to identify microorganisms that might be associated with the inflammation resulting from MAP infection. We also investigated the plausible correlations between the microbiome and the infectious doses analogous to environmental contamination by low and high shedding ruminants and age at which calves are vulnerable to MAP. A total of 33 Holstein calves were orally subjected to MAP (strain K10-like) at 5 distinct ages (2 weeks, 3, 6, 9 and 12 months) with a high (2x 5.109 CFU), low (2x 5.107 CFU) or no bacterial dose. Fecal and ileum digesta samples were collected at 17 months of age. DNA was extracted and V1-V3 regions of bacterial 16S rRNA genes were amplified and subjected to Roche 454 pyrosequencing. Sequences were converted to categorical data using a standard taxonomy by comparison to the Silva 16S rRNA database using mothur software package. Data were analyzed using generalized linear mixed-model methodology and partial least square discriminant analysis to identify genera that were most characteristic of treatment groups. The model’s goodness of fit and predictive value was evaluated by the R2Y and Q2 estimates, respectively. A total of 20 phyla were represented in the fecal compartment and 22 phyla in ileum digesta. If a phylum was present in all animals at 1% of population or higher it was considered to be a core phylum. Firmicutes, Proteobacteria and Bacteroidetes were core phyla in

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all compartments. Tenericutes were core to only fecal while Actinobacteria, TM7, Chrysiogenetes, and Cyanobacteria were core to only ileum digesta. In each compartment, core phyla comprised approximately 97% of the total microbial composition. Variation among populations of Firmicutes, Proteobacteria and Tenericutes of the fecal compartment were observed (P < 0.001). Among the bacteria included in the model, several genera including Carboxydibrachium spp. and Sphingomonas spp. were significantly associated with low MAP dosage, while Anaerophaga spp. and Ruminococcus spp. were significantly associated with high MAP dosage in the fecal compartment. In the ileum, several genera including Methylobacterium spp. and Cupriavidus spp. were significantly associated with control, Chrysiogenes spp. and Fangia spp. were significantly associated with low MAP dosage while Clostridium spp., and Oscillibacter spp. were significantly associated with high MAP dosage. R2Y and Q2 for fecal and ileum compartments were 0.517 and 0.165, and 0.841 and 0.226, respectively. Age at the time of infection was a discriminating factor as well. The fecal microbiome of MAP-infected animals at either dose were significantly distinct from other groups and could become the basis for a new JD diagnostics.

265B Effects of an ecological engineered oxygenation event on the microbial community structure in an anoxic fjord in western Sweden Michael Forth*1, Bengt Liljebladh2, Anders Stigebrandt2, Per Hall3, Alexander Treusch1 1University of Southern Denmark, Institute of Biology, Denmark, 2University of Gothenburg, Department of Earth Science, Sweden, 3University of Gothenburg, Department of Chemistry, Sweden

The availability of oxygen has a high influence on the diversity of communities and the distribution of organisms in pelagic ecosystems. Hypoxic or anoxic conditions caused e.g. by stratification lead to reduced habitats for oxygen depending eukaryotic and prokaryotic life. In recent years, oxygen depleted bodies of water are becoming more common. It is expected that in the near future anthropogenic influences like e.g. climate change and agriculture will intensify this problem. Recently, more effort has been put into the restoration of hypoxic habitats. The Baltic deep-water OXygenation (BOX) project proposed to introduce oxygen into the long-term hypoxic or anoxic bottom waters of the Baltic Sea by using wind driven pumps to generate artificial halocline and deep-water ventilation.

The Swedish Byfjorden is a stratified system with a lower water column and benthic zone that has been anoxic for a long time. In addition, freshwater from rivers support a brackish, well-oxygenated layer of surface water with lower salinity than the deeper layers, strengthening the stratification. Because of this, the Byfjorden is an ideal model system for the Baltic Sea. As a part of the BOX project, a pilot study to test the artificial oxygenation was initiated in 2009. A pump was installed in the Byfjorden in order to mix surface water into the deeper layers. This supplies oxygen, but also reduces the stratification which leads to increased frequency of natural inflows.

In this study, we monitored changes in microbial community structure in response to the oxygenation project in the Byfjorden. We analyzed water column samples from before, during and after the oxygenation as well as from control stations in two nearby fjords using a molecular microbial community profiling method. Our results show a shift in microbial community structure in response to the oxygenation event. Indicator species for hypoxic or anoxic conditions like e.g. species belonging to the SUP05 clade, declined in abundance during the oxygenation event and entirely vanished after the oxygenation was accomplished. In contrast, species like e.g SAR11 that indicate oxic conditions were detected not only in surface waters but also deep into the water column after the oxygenation. Here, we present the results of our study in the context of biogeochemical and hydrographical data to show the impact of the oxygenation on the bacterial community.

266B Bacterial endophytes of forest conifers: specificity and adaptive potential of an unexplored symbiosis Carolin Frank*, Alyssa Carrell, Emily Wilson University of California, Merced, United States

The relationship between forest trees and symbiotic bacteria called endophytes dwelling throughout their tissues is likely to be one of the world’s most wide-spread symbioses, yet it is largely unexplored. Research on agricultural crops has shown that some bacterial endophytes beneficially affect host development and growth, and provide stress protection. Bacterial endophytes of forest trees are also

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likely to play important roles in host biology, but to date, the ecological significance of this symbiosis is unknown. The aim of this study was to identify long-term and context-dependent associations between subalpine pine trees and endophytes. We wanted to know if specific symbionts with potentially important roles in host biology are shared across individual trees, or alternatively, if there is a high turnover of random 'visitors' with little importance in host biology. Using16S pyrosequencing to profile the endophytic community in needles of adult limber pine (Pinus flexilis), we compared the bacterial community within and across individual trees, and across geographic locations. We found that there is high consistency in the bacterial needle community across samples. All trees are dominated by bacteria belonging to the genus Gluconacetobacter, pointing to a adaptive and specific relationship between P. flexilis and these bacteria. Gluconacetobacter and related genera are capable of N2-fixation in the rhizosphere (the area surrounding plant roots) and as endophytes of crops such as sugarcane and coffee. Subalpine forest ecosystems have naturally low N availability due to slow turnover of organic nitrogen. To our knowledge, the possibility of endophytic N2 fixation in this ecosystem has not been explored. A capacity to fix N2 would explain the high relative abundance of Gluconacetobacter sp. in all limber pine samples.

267B Development of rapid technologies for the assessment of biodegradation potential in contaminated groundwater using gene array technologies Alessandra Frau*1, M. J. Larkin2, L. Kulakov2, C. Allen2, A. Kulakova2, B. Mc Polin3, R. Doherty3 1Queen's University Belfast, United Kingdom, 2Queen's University Belfast - School of Biological Sciences, United Kingdom, 3Queen's University Belfast - School of Planning, Architecture and Civil Engineering, United Kingdom

Bioremediation is the use of micro-organisms to restore contaminated environments and has the potential to restore these environments inexpensively and effectively. However to implement and monitor a viable bioremediation strategy information is required regarding the species inhabiting the microbial community, its dynamics and metabolic activities. In the last decade the use of molecular biology methodology has greatly improved our understanding of this approach. In this context the aim is to assess the microbial community and the likely biodegradative genes and pathways in groundwater samples contaminated by hydrocarbons. Information obtained through a community study will be used to construct and validate a DNA microarray, tool which allows the rapid detection of key micro-organisms able to degrade contaminants and to monitor the biodegradation rate. The study involves the groundwater analysis from two sites contaminated by hydrocarbons. Each involves analysis of water from boreholes: upstream, within the contaminated area and downstream the contamination.

Samples were analysed using three approaches. Firstly, molecular biological methods using PCR (16s rRNA gene and functional gene amplification, DGGE, qPCR) and sequencing using a multiplex amplicon pyrosequencing method. Secondly a classical microbiological method using viable counts using different growth conditions. Lastly evaluation of pH, O2 concentration, redox potential and temperature, and the GC/MS analysis to assess pollutants concentration. The PCR based methods and the DGGE have led to an evaluation of the community dynamics at different time points over one year. Functional gene amplification has led to the assessment of genes involved in the aerobic and anaerobic degradation of BTEX, PAHs and alkanes. The data obtained through these amplifications, combined with the chemical parameters, has yielded information about the likely dominant pathways (aerobic and anaerobic) and dominant taxa. In progress are the qPCR and metagenomic sequencing analyses, through 454 sequencing of tagged amplicons from multiple samples. This approach allows a rapid and deep sequence coverage, to obtain high quality reads and to minimize the noise.

In conclusion comparing the DGGE, the functional genes amplification and the viable count results we have obtained an overview of the microbial community in our sites over time. We will combine this data with the metagenomic sequences and the qPCR analyses to confirm our hypothesis about the sites and to develop suitable microarray probes.

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268B Pyrosequencing based assessment of bacterial and fungal community compositions in compacted forest soils Beat Frey*1, Stefan Schmutz1, Franco Widmer2, Martin Hartmann1 1Swiss Federal Research Institute WSL, Switzerland, 2Agroscope Reckenholz Taenikon ART, Switzerland

Soil compaction has been recognized as a major disturbance associated with timber harvesting and we currently lack fundamental knowledge how these perturbations affect soil microbial diversity. In order to predict the ecological consequences of soil compaction as well as to evaluate the capacity of soils to recover, we assessed the resistance and resilience of soil microbial communities during these perturbation events.

Two unique trafficking experiments generating wheel tracks of different severities by adjusting a gradient of soil moisture contents have been used to explore the relationships between soil compaction and soil microbial diversity. The study comprised three independent wheel tracks per forest site with three levels of compaction (no, light, and severe) in each wheel track. Soil samples at 5cm soil depth were taken 30, 180, 360 and 1440 days after trafficking and metagenomic DNA was extracted from these samples. Microbial communities were analyzed by using massively parallel pyrosequencing of bacterial (16S rRNA) and fungal (ITS rRNA) markers. Two full 454-pyrosequencing runs (80 samples, bacteria and fungi) have been processed and yielded a total of 1'678'558 raw sequences.

Raw sequences were processed using an established bioinformatic pipeline, which yielded a total of 996'340 (6'227 ± 2'782 per sample) high-quality sequences for community analysis. These data allowed for characterizing phylogenetic components of microbial communities in compacted and adjacent undisturbed soils. Basidiomycota (52%), Ascomycota (29%), Proteobacteria (43%), and Acidobacteria (30%) were the predominant phyla in these soils. Soil compaction had a substantial significant impact on both bacterial and fungal community structures and no resilience was observed up to four years after the compaction even. The strongest effect was observed in the severely compacted wheel tracks where air and water conductivities were reduced permanently to 10% or even lower of the original conductivities of undisturbed soils. At a higher order level, abundance of Ascomycota, Firmicutes, and Deltaproteobacteria significantly increased in severely compacted soils, whereas Basidiomycota, as well as Alpha- and Gammaproteobacteria decreased. Groups like Actinobacteria and Acidobacteria were significantly more abundant in the intermediate compacted soils when compared to uncompacted or severely compacted soils.

The analysis of the bacterial and fungal community compositions in compacted soils reflected the modifications to their habitat and highlighted the link between soil physical disturbance and microbial community responses in forest soils. Our data allow for the identification of microbial groups characteristic of conditions generated by soil compaction and provide indicator species that can be monitored for measuring similar disturbances.

269B Spatial and temporal variations of bacterial community in a sewage polluted urban river: the Zenne River (Belgium) Tamara Garcia-Armisen*1, Ozgul Inceoglu1, Natacha Brion2, Adriana Anzil1, Pierre Servais1 1Ecologie des Systèmes Aquatiques / Université Libre de Bruxelles, Belgium, 2Analytische en Milieu Chemie/Vrije Universiteit Brussel, Belgium

Sewage contaminated rivers are ecosystems strongly disturbed by human activities. Sewage discharges bring high loads of organic and inorganic pollutants but also allochthonous microorganisms, which can influence the biogeochemical cycles and could represent a health risk for some water utilizations. Despite the importance of this kind of freshwater ecosystems, little is known about their bacterial community composition (BCC). Our objectives were to study the spatial and temporal variations of the BCC along a sewage polluted urban river. For this, 4 sampling campaigns were performed in 2010 in the Zenne River in Belgium. Seven stations along the river and the treated effluents of the two Brussel’s WWTPs, were sampled. The BCC was analysed using 16S rDNA pyrosequencing. In parallel, a large set of environmental parameters were measured: temperature, pH, O2, conductivity, suspended solids, nutrients, organic carbon, heavy metals, bacterial abundance and production.

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The analysis of the environmental parameters revealed quite extreme conditions in terms of organic matters loads, bacterial abundance and production and also highly variable situations over space and time related to sewage discharge. The BCC analysis revealed the richness of this ecosystem and also confirmed its complexity. In general, sewage pollution seems to have a globally positive impact on bacterial diversity and evenness. The richness (observed OTUs) and the bacterial diversity (Shannon index) of the different samples were compared (ANODEV and ANOVA, respectively). The results showed that the seasonality was the most important parameter in the observed variations. Multidimensional scaling (MDS) on BCC also confirmed this finding. Two major grouping was seen: winter/fall samples versus spring/summer samples. Various environmental parameters appeared to be associated with the grouping: temperature, oxygen concentration, nutrients, bacterial abundance and production.

Furthermore, sampling stations were split into three groups, the two samples collected before WWTP (upstream), the five located downstream of the WWTP and WWTPs effluent. ANOSIM analysis based on BCC of these groups revealed that upstream samples were significantly different from downstream and WWTP effluents, while no significant differences were found between WWTPs effluents and the downstream samples.

At high phylogenetic level, the most abundant classes in the Zenne River were: Betaproteobacteria, Flavobacteria, Actinobacteria, Gammaproteobacteria and Epsilonproteobacteria that cumulated 75% of the sequences. At genus level, the most abundant were: Flavobacterium, Arcobacter, Malikia Rhodoferax, Zoogloea and Tetrasphaera. Indicator species analysis was used to identify individual bacterial genera that were indicative of either WWTPs or river samples. More than 40% of the sequences corresponded to rare phylotypes that represented less than 1% of the sequences.

These results highlight the metabolic potential of this unique bacterial community resulting from the mix of river and WWTPs microbiota and also the high diversity and evenness in the Zenne waters. As previous studies demonstrated the capability WWTP’s bacteria to remain active once discharged in rivers and recent research demonstrated the importance of the initial evenness to preserve the functional stability of an ecosystem, one might expect that this kind ecosystems despite being polluted have an important capability to adapt and resist to environmental stress.

270B Variations of microbial abundance and diversity along a 260cm long peat core indicative of flooding in the earliest holocene in central China Linfeng Gong*, Wang Hongmei, Liu Qiao China University of Geosciences, China

Microbial lipids such as long chain unsaturated ketones and glycerol dialkyl glycerol tetraethers are widely used to reconstruct the ancient climate conditions. To date, however, the potential of microbial abundance and diversity as climate indicators is less addressed in geological records. Here, we investigated the diversity and distribution of bacteria and archaea, and functional genes of methanogen and methanotroph in a 260 cm depth core, DZK-03, from the Dajiuhu peatland (31°28′50″N, 110°00′90″E, 1757 m) in central China via 16S rDNA clone library and real-time PCR. Totally 10 phyla and 56 genera of bacteria were detected in the peat core. Proteobacteria dominates among the phylum with Burkholderia as the dominant genus below 15 cm while Acidobacteria predominates in the upper 15 cm of the core. Archaea is predominated by Crenarchaeota Group I including Group I.1b, I.1c, I.1d and Group I.3. Surprisingly, Group I.3 dominates in crenarchaeota Group I with an exception at the depth 15 cm dominated by Group I.1b. Bacterial diversity fluctuates downward the core and shows a negative correlation with the copy number ratio of archaea to bacteria (A/B) and the copy number ratio of methanogen mcrA gene to methanotroph pMOA gene (M/P). We found the lowest Shannon-index value of bacteria and enhanced values of A/B and M/P at ca. 10 kyrs B.P., corresponding to a depth of 190 cm. As methanogen lives in anaerobic conditions and methanotroph in aerobic conditions, the ratio of M/P could indicate the redox condition which is driven by the peatland water table. The highest M/P value observed at the depth of 190 cm indicates the predominance of anaerobic condition resulting from the elevated water table in the peatland. In combination with the index of lipid flux, carbon deposition flux and sporopollen data, we propose the Dajiuhu region might undergo the flooding which in turn results in the presence of anaerobic conditions favoring the thriving of methanogen at ca.10 kyrs B.P.. Our data imply that the microbial diversity and abundance in the peat core could serve as the potential index for palaeoclimate reconstruction.

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271B Deconstructing the Microbial Community Degrading Plant Material in a Wet, Tropical Forest Dara Goodheart*1, T.C. Hazen2, M.K. Firestone3 1UC Berkeley, USA, 2University of Tennessee, Knoxville, 3UC Berkeley

The lower montane tropical forest in the Luquillo Experimental Forest in Puerto Rico has the highest litter decomposition rate measured to date. This soil undergoes frequent oxic/anoxic fluctuations lasting from days to weeks, caused by rain events. The persistent fluctuating environmental condition in the Luquillo Experimental Forest creates two disparate conditions (oxic and anoxic) under which distinct metabolic processes function. The persistent fluctuation in the environmental conditions could enable the rapid litter decomposition by coupling the oxic and anoxic decomposition processes.

The goals of this research are to: i) determine the abundance and identity of the bacterial and fungal communities degrading different components of plant material (whole lignocellulose, cellulose and lignin) and ii) assess the metabolic potential of the microbial community under each environmental condition and substrate in a wet, tropical forest soil.

To address these goals, soil from the Luquillo Experimental Forest was used in microcosm studies with 13C-labeled plant material added (either Avena barbata -whole lignocellulose analog, cellulose, or vanillin-lignin analog) under either continuous oxic, continuous anoxic or four day oxic/anoxic fluctuation. Gas samples were taken and triplicate Sample (with substrate) and Control (no substrate) soil jars were destructively sampled over time. DNA was extracted and used for qPCR of the bacterial 16S and the fungal ITS, 16S pyrotags and GeoChip 4 analyses.

As expected, cumulative CO2 and CH4 were greatest in the oxic and anoxic conditions, respectively, for each substrate. The cumulative CO2 and CH4 from the fluctuating condition of each substrate were similar to the cumulative CO2 and CH4 of the oxic condition.

Fungal abundance increased under oxic conditions and dramatically decreased under anoxic conditions with cellulose as the substrate, suggesting fungi are the main agents of decomposition under oxic conditions for cellulose in this soil. The abundance of bacteria does not change in response to environmental condition. However, 16Spyrotag data shows that the structure of the bacterial community altered in response to the environmental condition and substrate.

We used the GeoChip to assess the functional capacity of the microbial community under each environmental condition and substrate. The relative abundances of carbon degradation genes altered in response to environmental condition and substrate. In particular, cellulases and hemicellulases showed the greatest changes in relative abundance due to the presence of substrate and environmental conditions, respectively.

The abundance, structure and metabolic capacity of the microbial community altered in response to both the environmental conditions and the substrate. This dynamic response could drive the observed rapid decomposition rate in this wet, tropical forest soil.

272B Integrated assessment of freshwater streams associated with wastewater treatment plant discharge: microbiological and ecotoxicological approaches Adrienne Gregg*, Hai Doan, Anupama Kumar CSIRO Land and Water, Australia

The discharge of effluent from wastewater treatment plants can result in detrimental effects on the health of aquatic ecosystems. In Australia, treated effluent discharged from wastewater treatment plants can make up to 100% of the flow in many rivers, particularly during draught. Currently, we have a limited understanding of the impact of micro-pollutants from wastewater treatment plant effluents on the aquatic ecosystem. Assessment of river health is widely based on ecotoxicological approaches using fish and other macro-invertebrates as bioindicators. Environmental disturbances also influence the diversity and function of microbial communities therefore microorganisms may be used as bioindicators to assess the condition of aquatic ecosystems.

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The aim of this study was to use an integrated ecotoxicological, bioanalytical and microbiological approach to assess the impact on streams receiving wastewater treatment plant discharges. Two diverse environmental scenarios were selected: Wodonga West treatment facility,(New South Wales), located in a temperate climate where treated effluent was highly diluted within the receiving environment; and Wetalla treatment plant, Toowoomba (Queensland), located in a sub-tropical climate where the treated effluent was minimally diluted. Five in-stream sediment samples were collected approximately 500 m upstream of the treatment plant outfall (inferred as a reference site), at outfall and then progressively at 10, 100 and 1000 m downstream of the outfall. Conventional ecotoxicological methods were employed, which include in vitro assays based on yeast estrogen and androgen screens and whole-sediment bioassays using midges (Chironomus tepperi). The bacterial community structure and composition in the sediments was evaluated using PCR-DGGE coupled with the use of Shannon index and 16S rDNA clone analysis.

Site W exhibited a significant shift (pese results highlight that the loading of carbon and nitrogen into the creek from the outfall can affect major responses in bacterial abundance and community structure. A combination of effective treatment technologies employed by the treatment plants and the dilution based stream attenuation can provide a protective barrier for potential detrimental effects on the aquatic ecosystems receiving wastewater treatment plant discharges. In this study, the inclusion of soil microbial approaches to traditional ecotoxicological assessment has provided an additional line of evidence to successfully establish the influence of the discharged treated effluent in the receiving environment. We suggest an integrated approach combining advanced methods of ecotoxicology and environmental microbiology to achieve an improved understanding of the ecology of freshwater sediments and for a more realistic risk assessment of contaminated sediments in freshwater environments.

273B Gut bacterial communities in the giant land snail Achatina fulica and their modification by sugarcane-based diet M. Angela B. Grieco*1, Alexander M. Cardoso2, Janaína J.V. Cavalcante2, Ricardo P. Vieira3, Joyce L. Lima3, Maysa , M. Clementino4, Ana Tereza R. Vasconcelos5, Eloi S. Garcia2, Wanderley de Souza2, Rodolpho M. Albano6, Orlando B. Martins3 1Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brazil, 2Diretoria de Programas, Instituto Nacional de Metrologia, Qualidade e Tecnologia, Brazil, 3Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil, 4Instituto Nacional de Controle da Qualidade em Saúde, Fundação Oswaldo Cruz, Brazil, 5Laboratório Nacional de Computação Científica, Brazil, 6Departamento de Bioquímica, Universidade do Estado do Rio de Janeiro, Brazil

The invasive land snail Achatina fulica is one of the most damaging agricultural pests worldwide representing a potentially serious threat to natural ecosystems and human health. This species is known to carry parasites and harbors a dense and metabolically active microbial community; however, little is known about its diversity and composition. Here, we assessed for the first time the complexity of bacterial communities occurring in the digestive tracts of field-collected snails (FC) by using culture-independent molecular analysis. Crop and intestinal bacteria in FC were then compared to those from groups of snails that were reared in the laboratory (RL) on a sugarcane-based diet. Most of the sequences recovered were novel and related to those reported for herbivorous gut. Changes in relative abundance of Bacteroidetes and Firmicutes were observed when the snails were fed a high-sugar diet, suggesting that the snail gut microbiota can influence the energy balance equation. Furthermore, this study represents a first step in gaining a better understanding of snail microbiota and shows that its holobiont is a complex system containing diverse, abundant and active microbial communities.

274B Microbial activity and profile of bacterial communities in soil grown with sugarcane as affected by straw cover and soil moisture Régia Gualter*1, Vitor Vargas1, Thiago Gumiere2, Pedro Andrade2, Fernando Andreote2, Heitor Cantarella3, Adriana Silveira3

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1Pós-Graduação em Agricultura Tropical e Subtropical, IAC, Brazil, 2Escola Superior de Agricultura “Luiz de Queiroz”, Brazil, 3Instituto Agronômico-IAC, Centro de Solos e Recursos Agroambientais, Brazil

Soil microbial community is very sensitive to changes and alterations that occur in their habitat, especially those associated with key functions such as decomposition and input of organic matter and nutrient cycling. In this sense the microorganisms are potential indicators of soil quality. After sugarcane is harvested without burning, a thick mulch of straw remains on the soil. Potential changes in soil microbiota based on factors such as straw and moisture have been little studied in sugarcane, which is grown in more than 5 Mha in the São Paulo State. The objective of this study was to evaluate the changes in activity and microbial community structure in soil previously cultivated with sugarcane as a function of straw and different soil moisture. The treatments were: without and with 16 Mg ha-1 of straw and four soil moisture (100, 75, 50 and 30% of the pore space ground), conducted in soil cylindrical columns, under lab conditions at 142 days. The experimental design was completely randomized with three replicates. The variables analyzed were: microbial biomass carbon, soil respiration, metabolic quotient and community structure of Bacteria using the denaturing gradient gel electrophoresis of polymerase chain reaction-amplified DNA (PCR-DGGE) of 16 S rRNA gene. The data were analyzed using redundancy analysis (RDA), analysis of similarity (ANOSIM) and the Monte-Carlo test. Straw had a great affect as compared to soil moisture on the parameters evaluated. The PCR-DGGE revealed that the structures of bacterial communities were influence by straw cover and it was observed the formation of a gradient with the soil moisture, from higher to lower moisture. The similarity with straw cover was higher than without straw. The Shannon and Simpson’s index were similar for all treatments. The treatment with straw cover and 100% soil moisture showed the greater diversity (H= 3.758) and dominance (D=0.9756) indexes. The straw cover and soil moisture directly influenced the activity and the profile of bacterial communities.

275B Abundance and diversity of boxB, a diagnostic gene for aerobic benzoate catabolism, in a coastal norwegian fjord and computational diversity predictions in other metagenomes Christopher Gulvik*1, T. Chad Effler1, Reantha Pillay2, Alison Buchan1 1The University of Tennessee, United States, 2Farragut High School, United States

Aromatic compounds are abundant in terrestrial and marine systems. As such, microbial remineralization of these compounds is an important component of the global carbon cycle. The benzoyl-CoA oxidation (box) pathway is a recently described aerobic route for benzoate catabolism that has been experimentally identified in the soil betaproteobacterium Azoarcus evansii KB740 and the coastal marine alphaproteobacterium Sagitulla stellata E-37, a member of the abundant Roseobacter clade. However, the prevalence and genetic diversity of genes encoding this pathway in the environment is unknown. To address this gap in our knowledge, we have developed a degenerate qPCR primer set targeting the diagnostic epoxidase-encoding boxB gene and applied it to natural samples collected from a Norwegian fjord. Using this gene assay, abundances of boxB were found to range from 0.33-7.2% (expressed as percentage of bacterial 16S rRNA gene copies) in the fjord. Sequence analysis of clone library representatives suggests BoxB is strongly conserved at the amino acid level (74% identity). The majority of the clones (19/24) grouped within sequences derived from Alphaproteobacteria and 21% grouped within those from Betaproteobacteria. Because no other clones grouped outside of these two classes, the box pathway may be taxonomically constrained. In total, 67% of the clones grouped strongly within sequences derived from Roseobacter clade members. These data suggest roseobacters are the most abundant taxon in the fjord possessing this pathway. To extend the sequence analysis to other environmental systems, we developed a computational tool, tentatively entitled De-MetaST-BLAST, to probe publicly available metagenome sequences with the degenerate primer set. Paired with a local BLAST server, each predicted amplicon is retrieved from a read sequence. In this way, not only were we able to estimate diversity of box in other systems but also provide a tool for the validation of degenerate primer sets. The degenerate primer validation and query tool will be made publicly available August 2012 as freeware.

276B Using shotgun metagenomic data for less biased 16S rRNA gene community analysis Jiarong Guo*, James Cole, Titus Brown, James Tiedje Michigan State University, United States

Tuesday 21 August


Pyrosequencing of SSU rRNA genes amplified directly from environmental samples is now standard procedure for surveying microbial community structure. In contrast, metagenomic shotgun sequencing does not depend on gene-targeted primers and amplification and thus is not affected by primer bias or chimeras, respectively. However, it is difficult to detect and recover SSU rRNA genes from shotgun data and then to analyze them because of the number of sequences and the short lengths of the reads. The rrn operon including genes encoding SSU and LSU (Large Subunit) of rRNA genes can be found in all microbes and at varied copy numbers in different taxa. This relative abundance of the rrn operon means rRNA genes have high coverage in shotgun data. We compared the phylogenetic profiles of the SSU rRNA genes in pyrotag and shotgun sequencing data from the same soil samples. Pyrosequencing reads were processed using the Ribosomal Database Project Pyrosequencing Pipeline (RDP) and Mothur. SSU rRNA gene fragments in shotgun reads were detected by BLAST and HMMER and then classified by RDP's Classifier based on known SSU rRNA genes. Retrieved shotgun reads that aligned to the variable regions covered by the pyrotag reads (150bp in V7 and V8) were clustered into operational taxonomic units (OTU) and beta-diversity among samples was analyzed. We found that short reads aligned to the primer regions shared high sequence similarity with the primers used for pyrosequencing, but we also detected bases that were not covered by primers. In this way, traditional universal primers were re-evaluated and some taxa such as Verrucomicrobia not covered by the primers were found in increased abundance. At the phylum-level, community profiles were similar for pyrotag and shotgun data, suggesting that our method for identifying SSU rRNA gene fragments in shotgun data are effective. Beta-diversity by Principle Coordinate Analysis (PCoA) revealed the clustering pattern of samples with shotgun data and pyrosequencing data of V8 and V7 (regions pyrosequencing covers well) were similar and supports the use of shotgun metagenomic sequencing for improved 16S rRNA gene based community analysis.

277B Bacterial community of the fresh volcanic deposits on barren land, grassland, and woodland in the Island of Miyake, Japan, as revealed by pyrosequencing-based analysis of 16S rRNA genes Yong Guo*1, Reiko Fujimura2, Yoshinori Sato3, Wataru Suda4, Kenshiro Oshima4, Masahira Hattori4, Takashi Kamijo5, Hiroyuki Ohta2 1United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Japan, 2Ibaraki University, Japan, 3Independent Administrative Institution National Research Institute for Cultural Properties Tokyo, Japan, 4The University of Tokyo, Japan, 5University of Tsukuba, Japan

A number of studies indicated that microorganisms are the first biological pioneers in newly deposited mineral volcanic substrates (i.e., lava, tephra, and volcanic ash), but the information about the effects of the subsequent vegetation succession on the early soil microbial community is still limited. This study aimed to analyze the microbial communities of volcanic ash deposits derived from the 2000 eruption of Mount Oyama, along an elevational transect representing barren land (B: altitude, 547 m), grassland (G: 455 m), and woodland (W: 386 m) on the Island of Miyake (Miyake-jima), Japan. The fresh volcanic deposits (C1) and the buried soil (2A) below the deposits were separately collected from all the sites in July, 2009, and in September, 2011. All samples were moderately acidic (pH, 4.2-4.7) and the C1 deposits contained trace amounts of carbon and nitrogen, while the 2A soils contained 5-10 % of carbon and 0.4-0.8% of nitrogen. A PCR-based 454-pyrosequencing of 16S rRNA gene was employed to compare the bacterial communities of the C1 deposits and the 2A soils. A total of 149,382 effective sequences of 16S rRNA gene V1-V2 region were recovered from the 12 samples. These sequences revealed 224 to 466 of operational taxonomic units (OTUs) at species level in each sample using MG-RAST platform. Proteobacteira was the most abundant phylum in all samples, accounting for 20-52% of total effective sequences. The other dominant phyla were Cyanobacteria (C1, 12-22%; 2A, 1-14%), Actinobacteria (C1, 11-17%; 2A, 18-28 %) and Firmicutes (C1, 4-11%; 2A, 6-18%), followed by other 2 major phyla (average abundance > 1%), including Acidobacteria (C1, 0.8-2.8%; 2A, 1.2-10.8%) and Bacteroidetes (C1, 0.2-1.0%; 2A, 0.3-2.0%). Principal coordinate analysis (PCoA) based on Ribosomal Database Project (RDP) taxa abundances showed that the bacterial communities in the six C1 deposits were clearly different from each other, but the differences in the six 2A soils were relatively small. These results suggested that the structure of early bacterial community in fresh volcanic deposits was influenced by the above-ground vegetation type.

278B Microbial communities in soils from different agricultural management systems revisited: A comparison of traditional and next-generation molecular tools Martin Hartmann1, Salome Schneider2, Beat Frey1, Franco Widmer*2

Tuesday 21 August


1Swiss Federal Research Institute WSL, Switzerland, 2Agroscope Reckenholz-Tänikon Research Station ART, Switzerland

The DOK long-term agricultural management experiment has been initiated in 1978 near Basel (Switzerland). It has been designed for the comparison of bio-dynamic, bio-organic, and conventional farming management practices and initial research focused on practical feasibility and productivity of organic farming. More recent emphasis has been given to farming-related effects on soil quality and assessment of microbiological soil characteristics. We have previously reported on a molecular ecology approach with PCR-based (T-RFLP and RISA) detection of bacterial and fungal ribosomal loci. These data have revealed highly consistent strong effects of long-term farmyard manure application and short-term crop cultivation; however, effects were much stronger for bacteria than for fungi. A classical sequencing approach yielded indications for specific bacterial groups that responded to the management influences, but did not allow for sufficient diversity coverage and sample throughput in order to determine robust management indicators. Recent developments in sequencing technologies stimulated a reassessment of this site at much higher resolution. Therefore, the same soil samples were reanalyzed with a massively parallel pyrosequencing approach targeting bacterial (16S) and fungal (ITS) ribosomal markers.

A total of 40 soil samples (bacteria and fungi) were processed in one full 454-pyrosequencing run, yielding a total of 1'008'970 raw sequences. Erroneous and other low-quality sequences were removed using an established bioinformatic pipeline, leaving 525'710 (6'571 ± 1'582 per sample) high-quality sequences for community analysis. Actinobacteria (31%), Proteobacteria (27%), and Acidobacteria (10%) as well as Ascomycota (53%), Basidiomycota (24%), and Zygomycota (10%), were the predominant phyla in these soils. The high-throughput sequencing analysis supported results from the previous profiling studies mentioned above and showed significant effects of farming systems on soil bacterial and fungal community structures. However, in contrast to earlier findings, treatments affected bacterial and fungal communities to a similar degree, indicating that the improved resolution gained by high-throughput sequencing of fungal markers identified effects that went undetected using traditional low-resolution genetic profiling approaches. Furthermore, whereas short-term crop effects on fungal communities were hardly detected in a previous study, the current data demonstrated the presence of a significant crop effect on their community structure, indicating specific association of fungal groups with the cultivated crops. In conclusion, high-throughput sequencing techniques appear to substantially increase resolution when studying microbial communities in the environment, and thus, are able to elucidate effects of environmental change that could go undetected using traditional molecular tools.

279B Characterization of microbial communities in a UASB reactor treating municipal sewage using sulfur-redox reaction process Masashi Hatamoto*1, Aida Azrina Azmi1, Shinya Ono1, Akinobu Nakamura1, Masanobu Takahashi2, Takashi Yamaguchi1 1Nagaoka University of Technology, Japan, 2Tohoku University, Japan

An anaerobic wastewater treatment process has the advantages of low energy consumption and low sludge production; among the anaerobic treatment process, an up-flow anaerobic sludge blanket (UASB) method has been represented as the core technology for an anaerobic wastewater treatment. The method has recently been applied to sewage treatment because such advantages, however treatment efficiency decrease under low-temperature condition. To overcome the limitation and to prevent the reduction of treatment efficiency, we have developed the sulfur-redox reaction process combined with the UASB and a down-flow hanging sponge (DHS) reactor system. In the sulfur-redox process, sulfate-reducing bacteria in the UASB reactor degraded organic matter. The sulfide, one of the COD compound, is oxidized to sulfate by sulfur-oxidizing bacteria in the aerobic DHS reactor. In our series of studies of the municipal sewage treatment using an UASB reactor with sulfur-redox reaction under low-temperature conditions, we found an attractive novel phenomenon where the anaerobic sulfur oxidation had occurred in the UASB reactor without the presence of oxygen, nitrite and nitrate as electron acceptors. The phenomena of anaerobic sulfur oxidation have not been well understood yet. Therefore, in this study we have investigated on microbial communities existed in the UASB reactor which probably enhanced the occurrence of anaerobic sulfur oxidation. We have operated the pilot scale UASB reactor treating municipal sewage, which containing the additional sulfate to enhance the sulfur-redox process. Sludge samples collected from the UASB reactor which showed the sulfur oxidation were used for cloning and terminal restriction fragment length

Tuesday 21 August


polymorphism (T-RFLP) analysis of 16S rRNA genes of the bacteria and archaea. The microbial community structures of the bacteria and archaea was determined and indicated that the genus Smithella and the bacteria belonging to recently described phylum Caldiserica were the dominant bacterial groups. Meanwhile, Methanosaeta spp. was the dominant group of the archaea. The results of the T-RFLP analysis yielded characteristic fingerprints for bacterial communities when the anaerobic sulfur oxidation was occurred, while the archaeal community structure yielded stable microbial community.

280B Comparative analysis of microbial diversity of Pockmark sediments of the Oslo Fjord Thomas Haverkamp*1, Øyvind Hammer2, Kjetill S. Jakobsen3 1University of Oslo, Norway, 2Natural History Museum, University of Oslo, Norway, 3Centre for Ecological and Evolutionary Synthesis, Dept. of Biology, University of Oslo, Norway

Pockmarks are geological features found in the seabed in many oceans. These crater-like depressions are thought to be the result of fluids or gas streaming through the sediments and are often associated with subsurface oil or gas reservoirs. A bathymetric survey in 2004/2005 discovered up to 500 pockmarks in the Oslo Fjord with a size ranging between 20-50 m width and 2-10 m deep. Although there were no obvious signs of gas and fluids being emitted from the pockmarks, some studies indicated that the pockmarks formed during the Holocene and were caused by groundwater flows. The lack of subsurface hydrocarbon sources make the Oslo Fjord Pockmarks interesting as oil/methane negative ecosystems to be used in comparative metagenomics. Nonetheless, the Oslo Fjord is human influenced and is a catchment area for fresh water runoff from the surrounding boreal forest and the Oslo Community area. It is therefore expected that the sediments in the Oslo Fjord contain polycyclic aromatic hydrocarbons (PAHs) related to human pollution. The microbial communities in the Oslo Fjord sediments are expected to break down the PAHs. Since the pockmarks could influence local water currents, it is assumed that the sedimentation rates within the pockmarks are different from outside thereby affecting the microbial communities present in the sediments.

The aim of this study is to describe the microbial communities in the oil/methane negative pockmarks of the Oslo Fjord and compare those with the surrounding sediments. This is the first study describing the microbial community composition of the Oslo Fjord pockmark sediments using 454 amplicon sequencing targeting both the V3 region of the 16S rRNA gene and Aromatic Ring-Hydroxylating Dioxygenase (ARHD) genes. The ARHD genes are involved in the breakdown of PAHs. We targeted the ARHD genes using the degenerate primer pair ac114F/ac596R which target ARHD genes in gram-negative bacteria. For this study we have collected sediment cores from three different pockmarks and two reference sites within the inner Oslo Fjord. We measured basic chemistry as well as polycyclic aromatic hydrocarbons from each of the cores at the surface of sediment and at 40 cm depth. For both depths we extracted DNA, and generated amplicon libraries of both target genes.

Here we describe the first results of our work and discus how our results compare to amplicon studies describing microbial communities in hydrocarbon related pockmarks and marine sediments.

281B Metabolic classification of "functional gene" amplicon libraries using HMM-denoising and filtering: Finding fhs from homoacetogens in next generation sequencing datasets Gemma Henderson*, Eric Altermann, Peter H. Janssen AgResearch Ltd., New Zealand

Microbes that belong to the same functional group or have similar phenotypes are often not phylogenetically related. Furthermore, microbes that are closely related may not be able to carry out the same functions. For these reasons, 16S rRNA gene amplicon sequencing is not always suitable to characterise functional microbial groups. Homoacetogens are metabolically and phylogenetically diverse microbes that can use the Wood-Ljungdahl pathway to convert CO2 and H2 to acetate, and can be considered a functional group. Formyltetrahydrofolate synthetase (FTHFS) plays a key role in the Wood-Ljungdahl pathway of homoacetogens and the gene that encodes it (fhs) has been used to assess the diversity of homoacetogens in environmental samples. This gene is, however, not limited to homoacetogens, which necessitates careful interpretation of gene sequences libraries.

The aim of this study was to develop tools to investigate bacteria with genes coding for formyltetrahydrofolate synthetase that are indicative of an ability to use hydrogen.

Tuesday 21 August


A database with over 3,000 aligned fhs sequences obtained from the non-redundant database maintained by NCBI was used to create a taxonomic framework for the analysis of high-throughput sequencing data. Fhs amplified from ten gastrointestinal tract samples and sequenced using titanium 454 sequencing were used to test and validate the taxonomic framework.

Initial taxonomic assignment showed that approximately 40% of sequences were not similar to known fhs sequences and could not be placed within the framework. Further investigation revealed that over 50% these unplaced sequences were non-specific amplicons that were unlike known fhs. Based on known fhs sequences, Hidden Markov Model (HMM) were developed to filter and remove non-specific amplicons. Unassigned sequences that remained after HMM filtering were similar to fhs and may stem from potentially new clades. The proportion of non-specific amplicons removed by HMM filtering from some samples was larger than in other samples, indicating that DNA from some samples is more likely to favour non-specific amplification of gene sequences. This may be because there are fewer genuine target genes in those samples. HMM filtering also facilitated the ranking of sequences based on similarity to those of known homoacetogens, estimating the likelihood that the protein coded by any given sequence was involved in hydrogen use rather than in pathways other than the Wood Ljungdahl pathway. Fhs diversity differed between samples, and sequences similar to those of known homoacetogens were more prevalent in some samples.

In summary, we developed and validated a pipeline to investigate bacteria with fhs that are indicative of an ability to utilise hydrogen. A taxonomic framework for fhs was developed that can be used to analyse high-throughput next generation pyrosequencing data. Fhs pyrosequencing data should be de-noised prior to analysis and the newly developed HMM models represent a quick and efficient means to accomplish this. This pipeline is suitable for use in large-scale trials and could be adapted to other functional genes of interest.

282B "High precision melting curve": a novel method for fast screening of environmental samples for changes in bacterial phylogenetic composition prior to deep sequencing Mathis Hjort Hjelmsø1, Louise Feld2, Lars Hestbjerg Hansen3, Carsten Suhr Jacobsen*4 1University of Copenhagen / Geological Survey of Denmark and Greenland, Department of Geochemistry, Denmark, 2Geological Survey of Denmark and Greenland, Department of Geochemistry, Denmark, 3University of Copenhagen, Faculty of Natural Sciences, Department of Biology, Denmark, 4Geological Survey of Denmark and Greenland, Department of Geochemistry / Center for Permafrost (CENPERM), Department of Geography and Geology, University of Copenhagen / University of Copenhagen, Faculty of Life Sciences, Institute of Environment, Denmark

When working with microbial diversity you often end up with a high number of samples. Therefore it is often informative to screen your samples for diversity prior to further analysis (e.g. deep sequencing), to identify interesting samples. Current gel based methods for doing this (e.g. DGGE) are slow, requires technical skill and the results are difficult to compare between runs and laboratories. We present a novel method for accurate and fast screening of phylogenetic composition of bacterial communities using "high precision melting curves" on phylogenetic marker genes (e.g. 16SrRNA genes). Our intension for developing the method is to be able to screen a large number of DNA or cDNA samples to select the most interesting samples for deep sequencing.

First we amplified our phylogenetic marker gene using quantitative Polymerase Chain Reaction (qPCR). This was followed by the production of a "high precision melting curve" with precise measurements of Relative Fluorescence Unit (RFU) with 0.1 °C increments. The RFU values were then normalized and the shape of the curve could be used as a measure of the phylogenetic composition of the respective sample. The 270 samples examined in this study, originated from agricultural soil under different pesticide and nitrogen source amendments. With the use of the "high precision melting curves" we could easily pinpoint treatments that were interesting for downstream applications. As a proof of concept some samples have been analyzed by Denaturing Gradient Gel Electrophoresis (DGGE) and 16s amplicon pyrosequencing to confirm the correlation between "high precision melting curves" and phylogenetic composition.

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In conclusion this novel screening method could be very valuable in identifying the phylogenetic composition of environmental samples early in the experimental setup, thus saving time and money on analyzing samples with no interest to the respective study.

283B Endophytic bacteria within the green siphonous seaweed Bryopsis: exploration of a partnership Joke Hollants*, Frederik Leliaert, Olivier De Clerck, Anne Willems Ghent University, Belgium

The term ‘symbiosis’ described in 1879 as ‘a beneficial alliance between two dissimilar organisms’ has been rewritten over the last decade. Symbiosis no longer defines a concept in biology; it basically ís biology. Symbioses are widespread covering diverse forms of relationships among multiple partners and support fundamentally important processes. Endosymbiosis with one symbiotic partner (endosymbiont) living intracellularly within the other (host), is the most intimate form of symbiosis. The host typically provides a nutrient-rich, sheltered environment for the endosymbiont. In turn, endosymbionts expand their hosts’ physiological capacities, enabling them to invade novel metabolic and ecological niches. In view of this, it has been suggested that interactions with bacterial endosymbionts play a role in the success of siphonous (that is single giant-cell) seaweeds in a range of marine habitats.

This study aims to explore the partnership between siphonous seaweeds and their intracellular bacterial communities, focusing on the green alga Bryopsis as host organism. The identity, diversity, uniqueness, stability, specificity, function, symbiotic nature and transmission modes of the endophytic bacterial communities within Bryopsis were examined by the full-cycle 16S rRNA gene approach. Statistical analyses were performed to identify the factors shaping the endobiotic bacterial community composition.

Results indicate Bryopsis harbours rather stable and taxonomically diverse endophytic communities composed of certain Bacteroidetes, Flavobacteriaceae, Labrenzia, Mycoplasma, Phyllobacteriaceae, Rhizobiaceae and Rickettsia species. Although the algal interior is repeatedly exposed to various other marine bacteria during wounding events, a highly specific mode of partner recognition seems to exist as Bryopsis selectively maintains and/or attracts the same bacteria globally. This specificity is confirmed by the clear distinctiveness of the intracellular bacterial communities from those occurring in the surrounding seawater, even while the endophytic Labrenzia, Phyllobacteriaceae and Rhizobiaceae phylotypes are closely related to free-living bacterial strains and could be isolated on artificial media. These latter endophytes seem true generalists which are laterally acquired from the environment by any Bryopsis host, regardless of host species, habitat and geography,to possibly fulfill functions such as nitrogen-fixation, photosynthesis and CO-oxidation. Also Mycoplasma, Rickettsia and Bacteroidetes bacteria appear to a greater or lesser extent horizontally transmitted to Bryopsis algae. The habitat-specific lateral acquisition of these obligate intracellular bacteria, however, more likely takes place from co-occurring hosts rather than from the surrounding seawater. Flavobacteriaceae species, on the other hand, are closely associated to Bryopsis as they seem vertically transmitted, obligate endosymbionts which show some degree of cospeciation. The unique, host specific presence of these bacteria within Bryopsis species from warm-temperate and tropical seas and their internal location in and/or in the close vicinity of algal chloroplasts, might indicate Flavobacteriaceae endosymbionts fulfil a significant role within the Bryopsis host.

Taken together, these results indicate that the Bryopsis-bacterial partnership fits into the universal eukaryote-prokaryote symbiosis picture in which a mix of one to two specialist and several generalist bacteria reside within a single host. These diverse and complex endosymbiotic communities might permit further expansion of host capabilities, suggesting that ‘it takes two to tango, but a whole crowd to stage dive’.

Tuesday 21 August


284B Simultaneous detection of diversity of bacterial 16S and IncP plasmids and merA genes in soil samples by amplicon pyrosequencing Peter Nikolai Holmsgaard*, Lars Hestbjerg Hansen, Søren Johannes Søren University of Copenhagen, Denmark

In order to pool different samples together for amplicon sequencing, sequences are normally distinguished by short sequence barcodes, MID tags, attached directly in front of the primer. This method is very effective at labeling the sequences, but it does however require that multiple identical primers with varying MIDs are kept in stock. To make the method more flexible we have designed two short universal linker sequences of 20 base pairs each called linker-U1 and linker-U2 that are added in front of the forward and reverse primers respectively. A library with all MIDs attached to the sequencing adapter in on end and a U-linker in the other can then be kept in stock and only one primer is needed for each desired sequence. This added flexibility means that fewer primers need to be kept in stock and new primers can easily be tested with whatever MIDs and adapters desired. Primers targeting different plasmid Inc groups can then be used and the ones yielding results can easily be tagged with MIDs. So far we have with success tested the U-linker system with primers specific for IncP-1, IncP-3, IncP-4 (IncQ), IncP-7, IncP-9, IncW, merA, and 16S on soil from a birch forest, heavy metal contaminated soil and sewage water. All of these primers are well tested and have been used extensively for many years.

In addition, we have then used the primers and our newly developed U-linker system to study the diversity of both bacteria and IncP plasmids in a so called biofilter. The biofilter is a box 20x1.2 meters filled with soil, straw and manure used to clean water left over from crop spraying and containing high concentrations of pesticides. Bacteria are capable of degrading many xenobiotic compounds like pesticides and the enzymes involved in these degradations are often encoded on plasmids. The bacterial and plasmid diversity is studied by amplicon pyrosequencing of the 16S v3-v4 region and primers targeting IncP-1, IncP-4 (IncQ), IncP-7 and IncP-9 plasmid groups. MIDs and sequencing adapters were attached to the sequences using the U-linker system.

285B Comparison of bacterial community structures in 13 wastewater treatment plants from two geographically distant cites in China using 16S rRNA gene-based pyrosequencing Anyi Hu*, Jibing He, Chang-Ping Yu Institute of Urban Environment, Chinese Academy of Sciences, China

Activated sludge (AS), a core component in wastewater treatment plants (WWTPs), harbors enormously diverse microbial community assemblages. The diversity and composition of microbial community play critical roles in maintaining the functional stability of ecosystems. Thus, a thorough understanding of the mechanisms governing the variation in microbial community in AS could provide a theoretical foundation for optimizing the AS process. Here, we used the 16S rRNA gene-based pyrosequencing technique to investigate bacterial communities in AS samples from 13 full-scale WWTPs from two geographically distant cites (Chongqing and Xiamen; the distance between two cities is approximately 1,200 km) in China, and obtained an average of 9,328 effective sequences per sample. Observed phylogenetic diversity was positively correlated with the average flow rate of WWTPs, providing evidence that the species-area relationship may be a fundamental ecological rule in microbial systems. Rank abundance curves analysis indicated that while the rare biosphere constituted more than 70% of the observed OTUs for each sample, nearly 90% of the total community was represented by the 200 most abundant OTUs. Furthermore, although 56% of 6,254 OTUs were confined to single sample, a few core OTUs belonged to Proteobacteria, Firmicutes, Actinobacteria, Bacterioidetes, Acidobacteria, Chloroflexi, Nitrospira and Synergistetes were identified in multiple samples. Most core OTUs could be assigned to respective family or genus such as Bradyrhizobiaceae, Sphingomonadaceae, Nitrospira, Rhodobacter, Tetrasphaera and Thauera which might play an important role in removing nutrients and containments. Multivariate statistical analyses indicated a heterogeneous distribution of the bacterial communities between samples from Chongqing and Xiamen driven by both local and regional factors, whereas different bacterial phyla or groups may closely associated with different local environmental variables. In addition, among the 91 pathogenic genera considered, 389 putative pathogenic sequences (0.32% of the total) affiliated with 18 genera were identified in AS samples including some globally important human pathogens such as Brucella, Clostridium, Mycobacterium, Shigella, Staphylococcus and Streptococcus, suggesting the health risk when constantly exposed to AS.

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287B Systematic design of 18S rDNA primers for assessing eukaryotic diversity Luisa Hugerth*1, Daniel Lundin2, Emilie Muller3, Laura Lebrung3, Hugo Roume3, Paul Wilmes3, Anders Andersson2 1Science for Life Laboratory, Sweden, 2Kungliga Tekniska Högskolan, Sweden, 3University of Luxemburg, Luxembourg

PCR amplification and sequencing of SSU rRNA genes directly from environmental samples has revealed an enormous microbial diversity, in all the three domains of life, and the recent introduction of high-throughput sequencing technologies has made it possible to assess this diversity in large-scale comparative studies. However, for PCR-based diversity surveys to be non-selective and phylogenetically informative, it is crucial to use optimal PCR primers. While for 16S rRNA, the best regions to sequence and primers to use have been rather well investigated, no systematic design of optimal 18S rRNA primers for eukaryotic diversity studies has yet been reported.

We have developed the program DeBug for finding the highest covering primer at every position within a sequence alignment, given a maximum allowed degeneracy, and applied this to design a set of broad-taxonomic range 18S PCR primers based on existing eukaryotic database sequences and their annotation. We simulated the phylogenetic information that each candidate primer pair would retrieve using various read lengths and types (single or paired) representing different next-generation sequencing technologies.

A primer pair targeting the V4 region performed best in these tests and was subsequently used to successfully amplify 18S rDNA from a range of environmental samples including seawater, fecal samples, and sludge. This pair was used for Illumina sequencing of 18S rDNA of a set of model organisms to assess the amplitude of sequencing errors that can be anticipated in Illumina-based 18S surveys.

288B The Structure of Microbial Community in the Artificial and Natural Wetland Using Pyrosequencing Data Yeoung Min Hwang*, Keun Sik Baik, Seong Chan Park, Han Na Choe, Sonny Ramos, Ju Sik Cho, Dong Cheol Seo, Chi Nam Seong Sunchon National University, South Korea

We studied the application of high-throughput pyrosequencing to investigate the bacterial community structure of artificial and natural wetland. DNAs were extracted directly from the sediments obtained from the artificial (AW; 7 sites) and natural (NW; 2 sites) wetlands, Republic of Korea. A total of 69,072 (AW, 52,208; NW, 16,804) sequence reads, with an average read length of 459 nucleotides from the V1-V3 region of the 16S rRNA gene were obtained; there were between 6,085 and 10,551 reads per sample. The sequences were classified according to their domains, Bacteria (AW, 50,527; NW, 16,607) and Eukaryotes (AW, 1,681; NW, 257). Proteobacteria (AW, 43.6%; NW, 38.2%) represents the predominant phylum, followed by Bacteroidetes (AW, 13.9%; NW, 19.0%), Acidobacteria (AW, 13.8%; NW, 6.5%), Chloroflexi (AW, 8.1%; NW, 8.4%), Actinobacteria (AW, 2.3%; NW, 6.5%), Firmicutes (AW, 1.9%; NW, 2.5%) and Nitrospirae (AW, 4.7%; NW, 2.2%). The genera Dechloromonas, Flavobacterium, EU335319_g, Arthrobacter, Clostridium and Nitrospira_g1, were the major constituents of each phylum described above in order, whose ratio varied among different samples. Other phyla accounted for less than 2.0% of the total bacterial sequences. Approximately 2,426 and 1,607 genera were identified in AW and NW, respectively. Principal component analysis showed that the structure of microbial communities of all sites except 2 AWs was similar.

289B Studying the gut microbiota using rainbow trout as a model: the dynamics of the microbiota changes after first feeding Hans-Christian Ingerslev*, Mette Boye, Inger Dalsgaard, Lone Madsen Technical University of Denmark, National Veterinary Institute, Denmark

Fish provide an excellent model for studying the gut microbiota. During the very first life stage of most fish species nutrition only comes from a yolk sac originating from the egg and not from external feed sources. During this larval phase the intestine is being formed and becomes functional as the amount of yolk sac protein is used up and feeding from external sources slowly takes over. Thus, studying the intestine in this developmental window is useful for examining the influence of feeding on the gut

Tuesday 21 August


microbiota dynamics. In this study we examined this in the commercially produced species rainbow trout (Oncorhynchus mykiss) using next-generation sequencing. The examined fish were reared in re-circulated freshwater supplied by groundwater and fed commercial fish feed. Intestines from the fish were collected at three different time points during the developmental stage between larvae and fry for subsequent DNA isolation; a) 2 days prior to first feeding; b) 26 and c) 49 days post first feeding, respectively. A 16S rDNA PCR targeting the V5 region using universal barcoded primers was run on DNA from each sample prior to sequencing on the Illumina HiSeq 2000 platform. Obtained sequences were then subsequently analysed using a recently developed software package and taxonomically classified against the GreenGenes database (Bion Meta software, genomics.dk, in prep).

A significant shift in the bacterial composition occurred as a result of feeding. At the larval stage the bacterial composition measured as amount of reads was dominated by bacteria from the phyla Bacteriodetes (46.1%), Proteobacteria (26.3%) and Firmicutes (13.4%). Following feeding the amount of Bacteriodetes decreased to 19% (26 days post feeding) and down to 6% (49 days post first feeding), while the amount of Proteobacteria increased to 33.3% and 48.5% after 26 and 49 days post first feeding, respectively. For the Firmicutes the amount of reads increased to 31% and 30% after 26 and 49 days post first-feeding, respectively. The dominant genus within Bacteriodetes at all sample points was Sediminibacterium, an environmental bacterium usually found in soil and sediments. For the Firmicutes, these mostly constituted of Clostridiales and Lactobacillales, while the Proteobacteria were more diverse and consisted mainly of bacteria from the gamma-class and to a lesser extent the alpha- and beta-classes. Since the yolk sac is considered sterile it is hypothesised that the bacterial flora in the intestine prior to first feeding reflects the one present in the surrounding water and that, later, bacteria in the feed colonise the gut and to a certain degree outcompete the flora initially established before feeding.

290B Microbial structure change in the groundwater samples from two boreholes at depths of -250m in Horonobe Underground Research Laboratory Kotaro Ise*1, Isao Nanjo1, Hiroki Amamiya1, Yuki Amano1, Teruki Iwatsuki1, Takahiro Asano2, Koji Kageyama3, Yoshito Sasaki1, Hideki Yoshikawa1 1Japan Atomic Energy Agency, Japan, 2CHUGAI TECHNOS Corporation, Japan, 3Gifu University, Japan

We characterized the bacterial and archaeal communities in the groundwater samples obtained from the two different boreholes (V250-M02 (horizontal direction) and V250-M03 (vertical direction)) excavated in the horizontal gallery at the depths of 250m in Horonobe Underground Research Laboratory (URL). Recently, it has become gradually clear that in deep subsurface environments there are quite variety of bacterial and archaeal species and they inhabit actively more than we thought in previous times. However, for the sake of the wide variety results of previous phylogenetic studies, there are no consensus opinions in deep subsurface microbial ecology in these URLs. Clone library and Q-PCR analysis of the bacterial and archaeal 16S rRNA gene was conducted. Characterized bacterial structures of M02 and M03 were quite different in spite of from almost same sampling points. The bacterial structures of M02 were mainly composed of β-Proteobacteria and γ-Proteobacteria, M03 were δ-Proteobacteria and Firmicutes. After about four months, the proportion of α-Proteobacteria and Firmicutes increased, instead β-Proteobacteria and γ-Proteobacteria decreased in M02. The distribution of M03 also changed, JS1 candidate and Chloroflexi increased, instead δ-Proteobacteria decreased. And this M03 bacterial community was quite similar to deep sea sediment microbial communities. The change of bacterial 16S rRNA gene number occurred connection with the community change, in M02 borehole from 1.8x107 to 8.1x105 (copies/ml), in M03 from 1.5x104 to 1.1x105 (copies/ml). From these results, in Horonobe URL, at first bacterial communities was dramatically affected by excavation of tunnels and boreholes, but after several periods passed that may become close to the original community and that is similar to deep sea sediment microbial communities. The microbial ecology in deep subsurface has been investigated in many times, these reported microbial diversities are mainly composed of some facultative anaerobic bacteria. However, from the results of this study, it is thought that results of microbial community structures should depend on the sample collected on which moments, and the tendency was observed that if the more oxidized condition was constructed the more abundant bacteria were detected. So, it should be considered that if there are oxidized conditions, the bacterial activities were overestimated than the actual subsurface environments.

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291B Similarity and composition of the bovine rumen bacterial community Elie Jami, Itzhak Mizrahi* Agricultural research organization, Volcani, Israel

The bovine rumen houses a complex microbiota which is responsible for cattle's remarkable ability to convert indigestible plant mass into food products. Despite this ecosystem's enormous significance for humans, the composition and similarity of bacterial communities across different animals and the possible presence of some bacterial taxa in all animals’ rumens have yet to be determined. We characterized the rumen bacterial populations of 16 individual lactating cows using tag amplicon pyrosequencing. Our data showed 51% similarity in bacterial taxa across samples when abundance and occurrence were analyzed using the Bray-Curtis metric.By adding taxon phylogeny to the analysisusing a weighted UniFrac metric, the similarity increased to 82%. We also counted 32 genera that are shared by all samples, exhibiting high variability in abundance across samples. Taken together, our results suggest a core microbiome in the bovine rumen. Furthermore, although the bacterial taxa may vary considerably between cow rumens, they appear to be phylogenetically related. This suggests that the functional requirement imposed by the rumen ecological niche selects taxa that potentially share similar genetic features.

292B Comparative analysis of microplankton from the surrounding seawater of two deep-water coral reefs in the Norwegian Sea Sigmund Jensen*1, Jessica Ray1, Michael D. J. Lynch2, Josh D. Neufeld2, Martin Hovland1 1University of Bergen, Norway, 2University of Waterloo, Canada

Deep-water corals live below the photic zone, in poorly understood dark and cold environments. Exogenous supplies of carbon and chemical energy available for the corals to build reefs are derived from a variety of sources, including plankton. To assess the metabolic potential of the reef-associated microbiome, we identified representatives from reef seawater proximal (centimetre distance) and distal (30 m above) microplankton using a combination of cultivation-dependent and cultivation-independent methods. Two coral reefs were sampled at 260 m depth in a hydrocarbon field of the Norwegian Sea in May 2011. Prolonged dark and low-temperature incubations of the reef proximal seawater, in a mineral medium solidified with gelrite and enriched with carbon dioxide and gaseous hydrocarbons or ammonium, stimulated growth and subsequent isolation of diverse Alpha- and Gammaproteobacteria associated predominantly with the Loktanella and Colwellia genera. Pyrosequencing of ribosomal RNA transcripts amplified directly from reef proximal seawater indicated that in situ activity was dominated by other Alpha- and Gammaproteobacteria, and Flavobacteria. Chloroplast-derived 16S rRNA suggested the coexistence of active Stramenopiles (diatoms), as supported by transcripts of 18S rRNA, in addition to Dinophyceae, Viridiplantae and Ciliophora. Greatest diversity in richness and evenness was seen for analysis performed on the rRNA genes. A large proportion of 16S rRNA gene clusters could not be reliably assigned to a taxonomy (i.e. 47-58% of taxa assigned to Order), suggesting notable phylogenetic novelty. There were distinct taxonomic and phylogenetic differences separating the sites (Bray-Curtis, Unifrac) but no clear indicator species associated with distal or proximal samples surrounding these deep-water coral reefs.

293B Changes in bacterial community degrading chlorinated compounds by Fenton reagent Eun Hea Jho1, Naresh Singhal2, Susan Turner2, Kyoungphile Nam*1 1Seoul National University, South Korea, 2University of Auckland, New Zealand

High concentrations of contaminants can inhibit their biodegradation by indigenous bacteria in groundwater. In such cases intermittent clean-up of hotspots using chemical processes may be used to complement biodegradation activity. The Fenton reagent is commonly used for groundwater treatment. It initiates a reaction that produces hydroxyl radical, a strong oxidant. As contaminated groundwaters tend to be anaerobic, the addition of oxidants can have an adverse effect on the structure and function of the established anaerobic bacterial community. This study investigates the effect of Fenton's reagent on an anaerobic bacterial consortium capable of degrading hexachloroethane (HCA) and tetrachloroethene (PCE), two common groundwater contaminants. Anaerobic batch tests were performed to assess the effect on HCA and PCE degradation following exposure of the bacterial consortium for different durations to Fenton reagent and the recovery time following exposure. Bacterial community composition and changes in community structure were determined using 16S rRNA gene sequencing and automated ribosomal intergenic spacer analysis

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(ARISA). The anaerobic bacterial consortium was dominated by three genera of Clostridia - Clostridium (56% of sequences), Sporacetigenium (33%), Peptostreptococcus (8%). The exposure to Fenton's reagent impacted on the bacterial community structure with downstream effects on HCA and PCE degradation. The number of peaks, which indicate the relative diversity of the consortium, decreased from 8 to 1 with 1 h exposure, while it decreased to only 5 with 2 d exposure. Such losses in the diversity are related to the complete loss of biodegradability of HCA or PCE after 1 h exposure, but the partial loss of biodegradability by the 2 d exposed consortium. The length of fragments at which the peaks were detected in the ARISA profile changed with exposure to Fenton reagent suggesting the changes in the structure. The structure changes were confirmed using the multidimensional scaling of the ARISA profiles. However, the microbial community structure may be partially restored and degradation re-established by allowing a suitable recovery period after Fenton reaction exposure. The application of Fenton reagent for remediation of chlorinated compounds has impacts on the indigenous bacterial community structures and their biodegradability. In this study, the changes in the biodegradability of indigenous bacterial consortium after exposure to Fenton reagent could be correlated to the changes in the community structures using the 16S rRNA gene sequencing together with ARISA and the findings contribute to better understanding of the applicability of Fenton reagent together with biodegradation.

294B Comparisons of red pepper effects on kimchi fermentation using pyrosequencing and 1H-NMR Hyun Mi Jin, Sang Hyeon Jeong*, Che Ok Jeon Chung-Ang University, South Korea

Kimchi fermentation usually relies upon the growth of naturally-occurring various heterofermentative lactic acid bacteria (LAB). Kimchi is usually processed with various seasonings, such as red pepper powder, garlic, ginger, green onion, fermented seafood (jeotgal), and salts, at low temperature. Among kimchi ingredients, we investigated effects of red pepper powder on kimchi fermentation because red pepper powder is the major ingredient. We prepared four types of kimchi samples using Chinese cabbage and radish with and without red pepper power and analyzed bacterial successions during kimchi fermentation using barcoded pyrosequencing analysis. As the results, bacterial operational taxonomic units of kimchi without red pepper powder decreased more quickly than those of kimch with red pepper powder. The bacterial classification showed that members of the genera Leuconostoc, Lactobacillus and Weissella were dominant in all kinds of kimchi and interestingly, relative abundance of the genus Weissella in kimchi with red pepper powder was higher than that without red pepper powder. Metabolite analysis using the 1H-NMR technique showed that kimchi without red pepper powder began to consume free sugars and produce organic acids, ethanol, and mannitol more quickly than kimchi with red pepper powder. The PCA analysis using all detected metabolites including carbohydrates, amino acids, organic acids, and others also showed that time-serial tendency of metabolites was clearly discriminated between kimchi with and without red pepper powder. This study suggests that red pepper powder has effects on kimchi microbial communities and metabolites during kimchi fermentation processes. Additionally in this study detailed information of kimchi fermentation will be discussed. This work was supported by the Technology Development Program for Agriculture and Forestry (TDPAF) of the Ministry for Agriculture, Forestry and Fisheries, Republic of Korea.

295B Seasonal dynamics of microbial communities in polycyclic aromatic hydrocarbon - contaminated sediments Gloria Johnston*1, Linda Farnham2, Daniel Lisko2, Carl Johnston2 1Kent State University, United States, 2Youngstown State University

Contaminated river sediments are a common worldwide problem. In the Mahoning River, Northeast Ohio, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, and heavy metals remain in the riverbank sediments along 51 km of the riparian corridor from 100 years of industrial pollution. These contaminated sediments are the main factor limiting aquatic life of the river.

This is the first study that uses molecular approaches to investigate the microbial ecology of these sediments. Our goals were to i) characterize the geochemistry, ii) analyze microbial community structure and diversity, and iii) to evaluate seasonal changes on microbial communities. Information on microbial dynamics within Mahoning River sediment will be useful in developing alternative remediation technology.

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Sediment samples were collected during winter, summer and fall of 2011. Triplicate cores were collected using stainless steel tubes, which were capped, placed on ice, and processed in the laboratory within 48 hrs. PAHs were determined by gas chromatography mass spectrometry. Metals were measured using sequential extraction followed by inductively coupled plasma emission. Organic matter, moisture, and pH were determined by standard methods. Sulfate was measured by ion chromatography. Total carbon and nitrogen were analyzed using a CN Analyzer. Total bacteria were enumerated by staining with 4',6-diamidino-2-phenylindole, dihydrochloride. DNA was extracted for Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis. DNA was also used to construct bacterial clone libraries.

Total PAHs ranged from 20 to 100 mg/kg. Organic matter content (~15%), moisture content (36- 61%) and pH (7.4- 7.8) were uniform along seasons. Sulfate concentrations were much higher during winter and fall (100 to 1400 μg/g) than in summer (4 to 23 μg/g). High concentrations of total iron (30 %) were consistent with previous studies of which 17 % corresponded to iron oxides. Other metals with high concentrations included aluminum, chromium, copper, manganese, lead and zinc. Total metal concentrations did not show seasonal variation, yet metal oxides did. High carbon to nitrogen ratios (~55:1) were constant along seasons indicating a nitrogen limiting system.

Contaminated riverbank sediments contained an average of ~3.0E+7 bacteria per gram over winter, summer and fall. T-RFLP analysis, using HaeIIIas the restriction enzyme, indicated that seasonality shaped the microbial community. Preliminary cloning and sequencing revealed members of δ-Proteobacteria, including Fe and Mn-reducing bacteria (GeobacterandLeptothrixsp.) and methanogens (SmithellaandSyntropussp.). Interestingly thermophilic bacteria (Thiobacillus aquaesulisandThermotogaesp.) were also found. Discriminate function analysis predicted temporal and spatial segregation for geochemical and T-RFLP data.

Our investigation indicates that T-RFLP-based diversity was segregated spatially and by season, indicating that microbial communities responded to environmental parameters. The presence of high amounts of total iron and both Fe and Mn-reducing bacteria will be further explored for potential anaerobic oxidation of PAHs. More diversity analysis is needed to improve the understanding of how environmental parameters and pollution may affect microbial communities.

296B Zooming in on Lactic Acid Bacteria during industrial malting Annelies Justé1, Sofie Malfliet2, Pieter Busschaert1, Sam Crauwels1, Luc De Cooman2, Guido Aerts2, Kris Willems1, Bart Lievens*1 1Laboratory for Process Microbial Ecology and Bioinspirational Management (PME&BIM), KU Leuven Association, Lessius Mechelen, Campus De Nayer, Belgium and Scientia Terrae Research Institute, B-2860 Sint-Katelijne-Waver, Belgium, 2Laboratory of Enzyme, Fermentation and Brewing Technology (EFBT), Consortium for Industrial Microbiology and Biotechnology (CIMB), Department of Microbial and Molecular Systems (M2S), LFoRCe, K.U.Leuven Association, KaHo Sint-Lieven, B-9000 Ghent, Belgium

Knowledge of microbial dynamics during industrial malting has been limited, partly because the conventional approaches often resulted in an incomplete picture of the microbial diversity. One of the most remarkable microbiological changes during malting observed with conventional plating methods is in the plate count numbers of the lactic acid bacteria (LABs), consisting of a group of interesting strains which may be exploited as starter cultures or food-grade biocontrol agents in the malting process. Nevertheless, so far not much is known about the structure of this particular LAB community during malting.

Therefore, malting samples from different industrial malting systems, i.e. from barley up to kilned malt, were analyzed for their microbial community of specific LABs using both culture-dependent and culture-independent methods, including classical plate counts on de Man Rogosa Sharpe (MRS) agar plates and Terminal Restriction Fragment Length Polymorphism (T-RFLP) and 454 pyrosequencing.

The LAB community appeared remarkably stable during the whole malting process with Weissella and Leuconostoc being the dominant genera. No significant differences could be detected between the LAB community in dry barley, in germinating barley or in malt. The predominant LABs occurring throughout the whole process were nearly all present in the barley itself. 454 pyrosequencing revealed

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the selective presence of some minor, but possibly interesting LABs like Pediococcus in a specific year of harvest. Comparison of 454 data from DNA extracts on the one hand and from de MRS agar plates on the other hand, pointed out that about 70% of the specified LABs were recovered on classical plates.

Altogether, these results create a scientific base for selection of specific LABs as starter cultures in industrial malting.

297B Molecular analysis of microbial communities in the hydrocarbon-bearing sediments of Lake Baikal Vitaly Kadnikov*1, Andrey Mardanov1, Tamara Zemskaya2, Nikolay Ravin1 1Centre “Bioengineering” RAS, Russian Federation, 2Limnological Institute, SB RAS, Russian Federation

The methane hydrates and natural oil seeps were discovered several years ago at the bottom of Lake Baikal, the largest and deepest freshwater basin in the world. We examined the microbial communities inhabiting the hydrate-bearing sediments and bitumen mounds associated with the oil seeps using pyrosequencing of 16S rRNA genes. Two sediment samples were used to assess microbial diversity at "Saint Petersburg" hydrate site located at 1400 meters depth, - the upper layer of sediments (0-1 cm depth), and the deeper sediments (85-95 cm depth) just above the methane hydrate. The shallow sediments were dominated by methanogenic archaea of the orders Methanomicrobiales and Methanosarcinales, while known ANME archaea, as well as sulfate-reducing bacteria, were not found. In the deeper sediments bacteria accounted for more then 70% of microbial community. Most of them were assigned to Chloroflexi, candidate division JS1 and Caldiserica. Methanogens were also found, but almost 90% of archaea represented different "unclutured" lineages. Overall our results are consistent with the microbiological origin of methane hydrates in Baikal. Organic matter produced in the water column reach the lakefloor and became processed by a consortium of different bacterial and archaeal heterotrophs and, in the absence of sulfate and other alternative electron acceptors, is finally converted to methane. Another kind of hydrocarbon-bearing sediments is represented by the bitumen mounds generated due to activity of natural oil seeps at the bottom of the lake. These regions harbour peculiar community of microorganisms and higher eukaryotes presumably driven by hydrocarbon degradation. We analyzed the microbial community of an active mound, comprising a dropper from which oil flows permanently. The bacterial community consists mostly of Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes. Most of these sequences were affiliated to uncultured lineages within these groups, and were most closely related to ones found in hydrocarbon containing environments. Cultured lineages of Alpha- and Betaproteobacteria were related to aerobic and nitrate-reducing bacteria involved in hydrocarbon degradation. Representatives of delta and gamma subclasses of Proteobacteria, Actinobacteria, Acidobacteria, Verrucomicrobia, Chlorobi, and TM7 phylum were found in minor amounts. Archaeal component included Methanosarcinales, Methanobacteriales and Methanomicrobiales, suggesting that methanogenesis is an important process in the anaerobic part of the bitumen mound. Overall the results of this study provide new information regarding previously uncharacterized ecosystems and show the value of high-throughput sequencing in the study of their microbial communities.

298B Temporal dynamics of the microbial comunity structure in dark ocean of the western north pacific Ryo Kaneko*1, Mario Uchimiya1, Hideki Fukuda1, Shoutaro Suzuki1, Hiroshi Ogawa1, Toshi Nagata1, Makio Honda2, Koji Hamasaki1 1The University of Tokyo / Atmosphere and Ocean Research Institute, Japan, 2Japan Agency for Marine-Earth Science and Technology, Japan

The dark water column of ocean, including the mesopelagic (200-1000 m depth) and bathypelagic (1000-4000 m depth) zones is one of the most extensive microbial biospheres on earth. Previous studies have suggested that the dark ocean's microbes play important roles in biogeochemical cycling at globally significant rates in the enormous dark ocean, yet little is known about diversity, lifestyle and metabolisms of microbial assemblages in the dark ocean. To expand our knowledge about unidentified microbial ecosystem in the dark ocean, we have investigated the temporal change of microbial assemblage in the subarctic and subtropical western North Pacific Ocean' s interiors. Meso- and Bathypelagic seawater samples (300 and 2000 m depth) were collected from the subarctic station K2

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(47°N, 160°E) and the subtropical station S1 (30°N, 145°E) in winter (February, 2010) and summer (July, 2011) on cruise of R/V Mirai. The physico-chemical parameters of water-column below 200 m depth in K2 and S1 were not shown marked seasonal change during January 2010 to July 2011. Four liters of seawater from each of two depth was filtered through 0.22-um pore Sterivex filter, and stored at -80°C upon arrival in the laboratory. Genomic DNA was extracted aseptically from filter samples, and 16S rRNA gene (16S rDNA) fragments were PCR amplified for the clone library analysis. The 16S rDNA sequences obtained were compared with current database sequences (NCBI) using BLASTn to determine phylogenetic relatedness, and were checked using several programs to remove chimeras. A total of 384 clones of 16S rDNA sequences yielded 15 phyla (91 OTUs at the 97% identity cutoff threshold) were retrieved from 16 clone libraries. Cluster analysis of 16S rDNA sequences indicated that microbial community structure in 300 and 2000 m depth water showed temporal, vertical and spatial change. The highest bacterial diversity (rarefaction analysis) and species richness (Chao1) was found in the summer of 2000 m depth of subtropical station S1; archaeal diversity and species richness was decreased with depth. The Alphaproteobacteria, Gammaproteobacteria, Deltaproteobakuteria and Thaumarchaeota were the common phylogenetic groups in all samples, and the prominent OTUs were member of the SAR11 clade of Alphaproteobacteria and Group I marine archaea. The relative abundance of OTUs relevant to SAR11 clade showed extensive temporal shift in 300 and 2000 m samples. In this study, we have revealed the unexpected dynamics of microbial community structure in meso- and bathypelagic realm of pelagic ocean. Our result contributes to the better understanding of the least explored dark ocean biosphere.

299B Microbial diversity and spatial distribution in deep subseafloor sediments of the Izena Cauldron, Okinawa Trough Sanae Kano*1, Yosuke Onishi2, Takashi Yoshida1, Hiroshi Ishida2, Nobuhiro Maeda2, Manabu Tatsuta3, Tetsuya Miwa3, Nobuhiro Goto4, Nobuyuki Okamoto5, Yoshihiko Sako1 1Kyoto University, Japan, 2The General Environmental Technos Co., Ltd, Japan, 3Japan Agency for Marine-Earth Science and Technology, Japan, 4Deep Ocean Resources Development Co., Ltd, Japan, 5Japan Oil, Gas and Metals National, Japan

The Okinawa Trough is a back-arc basin of the East China Sea in Japan. In the east side of mid-Okinawa Trough, a notable geological feature of Izena Cauldron has been found and identified type as sulphide deposit. Buoyant fluids released from the seafloor at 1,400 m depth contains CO2 (86%), H2S (3%), and residual gas (CH4+H2). It is seemed that the composition of the microbial community at the inside field of the cauldron would differ to that at the outside field of the cauldron. However, little is known about the microbial community in this field. Microbial diversity from sediments in the Izena Cauldron at the Okinawa Trough was investigated using molecular biological approaches.

The nine cores were collected from five outside (IZ1-3, 7, 11) and four inside (IZ4-6, 9) of the cauldron at the Izena fields using a Multiple Corer. Archaeal and bacterial clone libraries of 16S rRNA genes from both inside and outside fields of the cauldron were constructed and analyzed 3,500 sequences.

Our analyses of the bacterial sequences revealed much higher diversity than archaeal diversity. The microbial community structure of the Izena fields at the phylum-level revealed no significant difference between the inside and outside cauldron libraries. However, seven clusters, all of which were assumed to have sulfur metabolism, consisted of sequences from only inside fields of the cauldron in OTU clustering approach. Of these clusters, the largest-size cluster was the cluster E11 (N=9) of which representative sequence showed high similarity to an uncultured bacterium clone Out0bac49 belonging to Basal subgroup in Gammaproteobacteria and was phylogenetically closed to sulfur-oxidizing bacteria group. Further, we estimated the abundance of cluster E11 using quantitative PCR with a primer set specific to the cluster E11 16S rRNA gene. The copy numbers of its gene in inside the cauldron cores were higher in comparison to that in outside cauldron cores. The highest copy number of the cluster E11 was in the 2 cores (IZ-5 and -9) inside the cauldron. The presence of the bacteria belonging to the cluster E11 was notably not only inside the cauldron also outside the cauldron. Sulfide concentrations in subseafloor sediments of inside the cauldron were also higher than those of outside the cauldron. These results suggest that the bacteria belonging to the clusters, which are detected from only inside cauldron cores, are associated with sulfur-cycling in the core at the Izena Cauldron.

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300B Hydrazine synthase: a unique functional gene to study the biodiversity of anammox bacteria Boran Kartal*1, Harry R. Harhangi1, Mathilde Le Roy1, Theo van Alen1, Bao-lan Hu2, Susannah G. Tringe3, Zhe-Xue Quan4, Mike S.M. Jetten1, Huub J.M. Op den Camp1 1Radboud University Nijmegen, Netherlands, 2Zhejiang University, China, 3JGI, United States, 4Fudan University, China

Anaerobic ammonium-oxidizing (anammox) bacteria play an important role in the biogeochemical cycling of nitrogen. They derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. Several methods have been used to detect the presence and activity of anammox bacteria in the environment, including 16S rRNA gene based approaches. The use of the 16S rRNA gene to study biodiversity has the disadvantage that it is not directly related to the physiology of the target organism and that current primers do not completely capture the anammox diversity. Here we report the development of PCR primer sets targeting a subunit of the hydrazine synthase (hzsA), which represents a unique phylogenetic marker for anammox bacteria. The tested primers were able to retrieve hzsA gene sequences from anammox enrichment cultures, full-scale anammox waste water treatment systems and a variety of fresh water and marine environmental samples, covering all known anammox genera.

301B Cryopreservation techniques applied to the analysis of oceanic picoplankton diversity Masanobu Kawachi*, Mary-Helene Noel, Mikihide Demura National Institute for Environmental Studies, Japan

Cryopreservation techniques have been successfully developed in microbial culture collections or cell banks to achieve stable and long-term preservation of several culture strains. In the case of photosynthetic organisms, particularly fragile species are still hard to be cryopreserved and should be maintained by successive transfers into fresh medium. For such fragile species however, even if the cells lost their viability when cryopreservation is applied, they still keep their original shape and chlorophyll fluorescence at room temperature for several hours. In this study, we based our work from environmental specimens cryopreserved after sampling to obtain genetic and morphological information and some culture strains of cells from the pico-size fraction (less than 2 µm diameter). Biodiversity studies of pico-size fraction have indicated so far the existence of a large number of uncultured and unknown organisms, suggesting a very high biodiversity. Many of uncultured environmental sequences are now accumulated in gene bank but unfortunately without any biological information neither living strains.

Our strategy to work from cryopreserved environmental specimen may open new opportunities to avoid this bias since cryopreservation success of particular organisms would allow for repeated analysis and finally support and clarify the identity and the biodiversity of the particular group. We use a two-step freezing protocol for the cryopreservation of environmental specimens, i.e. concentrated samples with or without cryoprotectant is cooled with a rate of -1°C/min to -40°C by using a programmable freezer and then freezed rapidly to -196°C in liquid nitrogen. The best compromise of picoplankton cell count and viability from cryopreserved specimens was obtained by using DMSO at a final concentration of 5%. Enrichment treatment from cryopreserved specimens could confirm the survival of several species including picoplankton species that successfully grown and are now as culture strains.

Flow cytometry (FCM) is a powerful tool to analyze picoplankton community and the cell sorting capacity of such apparatus is an efficient way for both genetic analysis and establishing culture strain. FCM analysis of environmental species directly after sampling and after cryopreservation revealed that the cells population profile was retained, but the cell number drastically dropped to 50-80%. We checked the effect of the cryopreservation technique on environmental specimens to the cells morphology by using culture strains of representative picoplankton species and confirmed that the cell profile of most species was retained. In addition, we could confirm the good match of morphological observations with scanning electron microscope and genetic information of 18S rRNA gene clone library from the FCM sorted cells of the natural samples. We still need to improve the cryopreservation techniques as well as the culturing methods to increase the final viability of the environmental specimens from the cryopreservation. The present study clearly appeals for the effectiveness of a cryopreservation method applied to the analysis of the oceanic picoplankton diversity.

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302B Microbial community changes in response to chloramine residues in a lab-scale system Bal KC1, Arumugam Sathasivan1, Maneesha Ginige*2 1Curtin University, Australia, 2CSIRO, Australia

The maintenance of a “residual” of disinfectant until the water reaches people's homes is required in order to comply with the guidelines of the environmental protection agency. When chloramine is used as a disinfectant, managing its decay particularly once nitrification has been observed is challenging and often is a losing battle for the utilities. Managing chloramine decay prior to the onset of nitrification through effective control strategies is important and to date the control strategies developed based around nitrification has not been effective. This study aimed at developing a more holistic knowledge on how decaying chloramine impact microbial communities in chloraminated systems. Five lab-scale reactors (connected in series) were operated to simulate a full-scale chloraminated system. The reactors were operated under controlled conditions (initial chloramine residuals, pH and temperatures) and consistently produced water that had different nitrifying/chloramine residuals similar to full-scale chloraminated distribution systems. Culture independent molecular techniques were used to characterise the mixed microbial communities in the five reactors that maintained a chloramine residue of high to very low across reactor 1 to 5. The total bacterial numbers were estimated using flow cytometry and the abundance of specific bacterial groups were estimated using quantitative polymerase chain reactions. Total chlorine residuals maintained at 2.18 ± 0.1 mg/L in R-1 progressively decayed along the reactors and over a four-day retention period, the residual dropped down to 0.0± 0.03 mg/L at the time of reaching R-5. Of all reactors, a pronounced drop of chloramine and total ammonical nitrogen (TAN) residuals was observed in R-3, with a subsequent increase of nitrite and nitrate. This sudden change of conditions in R-3 compared to R-1 and R-2 indicated the onset of nitrification in this laboratory chloraminated system. NOx (nitrite+nitrate) production along the reactors R-1 to R-5 was 0.003, 0.016, 0.252, 0.120 and 0.070 mg-N/L, respectively. Additionally, the pH decrease from 8.10 in R-1 to 7.65 in R-5 also suggests nitrifying bacterial activities. The data of the study for the first time demonstrates the impact of chloramine residuals on different microbial communities and on the formation of nitrogenous compounds. Bacterial classes Solibacteres, Nitrospira, Sphingobacteria, Betaproteobacteria dominated at low chloramine residuals (below 0.65 mg/L) whereas Actinobacteria and Gammaproteobacteria dominated at higher chloramine residuals (1.65 to 2.18 mg/L). Prior to the onset of nitrification bacterial genera Pseudomonas, Methylobacterium and Sphingomonas were found to be dominant and Sphingomonas in particular rapidly increased with the onset of nitrification. Nitrosomonas urea andNitrosomonas oligotropha were detected once the chloramine residuals had dropped below 0.65 mg/L and nitrification alone was not sufficient to describe chloramine decay rates that were observed in these reactors. A soluble microbial product perhaps produced by microorganisms to resist chloramine is suggested to expedite chloramine decay alongside other physiochemical changes brought about by microorganisms such as nitrifiers. The findings of this study is expected to re-direct the focus in this area of research from nitrifiers to other heterotrophic bacteria, and could hold the key towards developing a control strategy to manage chloramine decay in chloraminated distribution systems.

303B Discovery of cellulose-disrupting enzymes from an avicel-adapted forest soil eukaryotic community by a metatranscriptomic approach Nobutada Kimura*, Kazuto Takasaki, Manabu Kanno, Yoichi Kamagata National Institute of Advanced Industrial Science and Technology (AIST), Japan

The majority of prokaryotes living in the environment have not yet been cultured. Likewise, environmental community analysis based upon 18S rRNA gene sequencing has also shown that a wide variety of eukaryotes such as fungi exist in environments and their large portions still remain uncultivated. Such diversity together with greater genome size than those of prokaryotes implies an enormous genetic and biological pool that can be explored for recovering novel genes, enzymes, entire metabolic pathways and their products. Here, we attempted to explore the genetic and functional diversity of eukaryotes based upon transcriptomic approach mainly targeting genes encoding glycoside hydrolase enzymes.

To discover the structural and functional novel glycoside hydrolase enzymes from soil eukaryotic communities that decompose cellulosic biomass, we amended the soil sample with cellulose and wheat bran for 6 days and incubated it as soil suspension, anticipating that those responsible genes are induced, expressed and upregulated. Transcripts of functional genes in a forest soil were analyzed using BLAST search according to the KEGG database with expected values above 1×10-8. Similarity

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search results against the UniprotKB and CAZy databases were referred to the functional prediction of cellulosic biomass-degrading enzymes.

Pyrosequencing of the avicel and wheat-amended soil cDNAs produced 56,084 putative protein-coding sequence (CDS) fragments, and the most dominant group of putative CDSs based on the taxonomic analysis was assigned to the domain Eukarya accounting for 99% of the total number of the putative CDSs. Of 9,449 eukaryotic CDSs whose functions could be categorized, approx 40% of the putative CDSs corresponded to metabolism-related genes including carbohydrate, amino acid, and energy metabolisms. Among the carbohydrate metabolism genes, 129 sequences encoded glycoside hydrolase enzymes with 47 sequences being putative cellulases belonging to 13 GH families. Moreover, 35 sequences show low amino acid sequence similarity (less than 70%) to any known cellulolytic enzyme-coding sequences. To characterize the glycoside hydrolase enzyme, we synthesized FX003685 gene with codon optimization for heterologous expression in Escherichia coli, which was shown to be very similar to the structure of plant expansins, and observed stimulation for cellulase activity on avicel degradation. This study demonstrate that microbial communities adapt to avicel and wheat decomposition and that novel genes can be identified from complex metatranscriptomic sequence data, synthesized and expressed resulting in active enzyme.

Metatranscriptomic approaches demonstrated unprecedented access to the genetic information of eukaryote with respect to biomass conversion processes. Cloning of the gene fragments encoding carbohydrate metabolic enzymes is currently under way to get more information of these enzymes. These approaches may provide more understanding of functions and diversity of carbohydrate metabolic enzymes and may lead to promote the application of these enzymes in decomposition of cellulosic biomass.

304B Investigating the etiology of bovine digital dermatitis by a combination of 16S rRNA gene analysis and fluorescence in situ hybridization Kirstine Klitgaard*1, Marianne Rasmussen2, Nynne Capion3, Mette Boye2, Tim K Jensen2 1National Veterinary Institute, Technical University of Denmark, Denmark, 2Technical University of Denmark, National Veterinary Institute, Denmark, 3University of Copenhagen, Faculty of Life Science, Department of Large Animal Science, Denmark

Bovine digital dermatitis, the cause of lameness and wasting in cattle, was first reported in 1974. Today, this disease has considerable negative effects on animal welfare and production economy in many parts of the world. A bacterial etiology of digital dermatitis is now well documented, and the current view on this disease points towards a complicated etiology involving co-infection of more than one, and probably multiple species belonging to the genus Treponema. Still, the pathogenic role of each of the digital dermatitis-associated phylotypes remains unclear. The aim of this investigation was to obtain a better understanding of digital dermatitis in general, including possible predisposing skin alternations and the role of the bacteria Dichelobacter nodosus. Finally, we wanted to determine if any Treponema phylotypes could be singled out as having a particularly prominent role in the etiology of the disease. Here, a PCR-based approach targeting the 16S rRNA gene, along with fluorescence in situ hybridization, was used to determine the prevalence and diversity of 17 Treponema phylotypes in 85 digital dermatitis lesions from six Danish dairy herds as well as additional biopsies of healthy skin and previously examined digital dermatitis lesions. All skin samples were evaluated histopathologically for possible predisposing abnormalities. Furthermore, fluorescence in situ hybridization tests for Fusobacterium necrophorum and D. nodosus were applied. lesions revealed intermingled infections with multiple Treponema phylotypes (mean > 7). In six herds, the mean number of phylotypes identified varied between 12 and 15. D. nodosus was present in forty-nine (51%) of the lesions and in three of the apparently healthy skin samples. One “healthy” sample also contained Treponema spp. and D. nodosus, and was histopathologically categorized as subclinical digital dermatitis. We propose that external noxious stimuli allow D. nodosus to break down the epidermal barrier creating a suitable environment for the secondary invaders, Treponema species, which gradually take over the infection site. The variety and different distributions of treponemes in the digital dermatitis lesions observed in this study, suggest that most of the Treponema phylotypes have the potential to be pathogenic.

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305B Analysis of microbial community functions involved in effective and eco-friendly industrial tannery wastewater treatment Sung-Cheol Koh*1, Kalu Ibe Ekpeghere1, Shin-Young Ha1, Soo-Hyeon Kim1, Hong-Gi Kim2, In-Soo Kim1, Bong-Keun Song3 1Korea Maritime University, South Korea, 2BM Company, South Korea, 3University of North Carolina, South Korea

This study was to indentify microbial populations involved in the industrial treatment of tannery wastewater using a microbial consortium (BM-S-1) and their specific roles during the treatment process. COD, T-N, T-P and Cr were monitored for water quality. Microbial population dynamics and functions were monitored using pyrosequencing and real-time quantitative PCR (RT-qPCR) analysis. The COD, T-N and T-P removal efficiencies were more than 97%, 72% and 98%, respectively and the chromium removal efficiency was 89%. Pyrosequencing data from different stages of the treatment processes revealed that the microbial community profiles of primary aeration, secondary aeration and sludge digestion were generally similar to each other, but not to the buffering stage. The most dominant species in all the stages were Brachydomonas sp. and other denitrifiers (6-37%) except the buffering stage. Cr (VI) reducing bacteria were dominant in the buffering and secondary aeration. The population density as revealed in the gene copies (nosZ) monitored by real-time quantitative PCR was found to vary at the different stages in decreasing order from the buffering stage to the sludge digestion. This technology can also be applied to the treatment of wastewaters carrying dyes and detergents.

306B Deep Ocean microbial communities in the South Adriatic Pit Marino Korlević*1, Danny Ionescu2, Paolo Paliaga1, Tina Šilović1, Rudolf Amann2, Mirjana Najdek1, Sandi Orlić1 1Ruđer Bošković Institute Center for Marine Research, Croatia, 2Max Planck Institute for Marine Microbiology, Germany

The Adriatic Sea is a semi enclosed basin in the Northeastern Mediterranean with a shelf that extends from its northernmost part to the Split Gargano transect. Its deeper southern part is characterized by the circular, 1243 m deep South Adriatic Pit (SAP). Adriatic ecosystems are influenced by regular water exchange with the Eastern Mediterranean through the strait of Otranto. Levantine Intermediate water and Ionian Surface Water flow into the Adriatic along its eastern border. The volume of this flow is greater in winter but changes from year to year depending on climate oscillations that occur from the Adriatic to the Southeast Mediterranean. In this study we used pyrosequencing of the 16S rRNA gene and DGGE to study the water column above the SAP in comparision to a close station (300 m depth). The total number of bacteria at station P1200 was between 2.6x105 at surface and 6.7x104 at the bottom with a peak of 3.3x105 at 75-100 m. At the shallower P300 station the change in bacterial counts was lower raging from 1.8x105 at the surface to 1.1x105 at 200 m. Pycophytoplankton counts using flow cytometry showed a Prochlorococcus peak at 100m depth in both P1200 and P300. Synecococcus peak was at 10 m in both stations and another peak at 20 m at P300 and 75 m at P1200. DGGE analysis of the P1200 samples showed 4 distinct communities: aurface (0 and 10 m), shallow deep (75, 100 and 200 m), intermediate depth (400, 600 and 800 m) and deep (1000 and 1200 m). A similar pattern was obtained by cluster analysis of the pyrosequencing data. Samples form P300 cluster together with those from similar depths from P1200 suggesting that distance from the surface is a significant factor in determing the community within the photic zone (300 m). Taxonomic analysis of the pyrosequnece data showed a depth related dsitribution of the various phylogentic groups. Intrestingly, some of these characteristics are typical to the deep ocean. Flavobacteria were present only in the upper 100 m while Deferribacteres, Acidobacteria and the Sar202 clade (Chloroflexii) were detected in significant numbers only below 200 m. The Alphaproteobacteria class was detected in high abundance throughout the water column; however, the Rhodobacteraceae and the SAR116 clade were more abundant in the upper 100 m while the Rodospirillaceae were frequent only in deeper waters. Wthin the Sar11 clade some groups where equally abundant at all depths while other showed clear preference to shallow deep waters. The Sar324 clade, found below 200 m, is the dominant group within the Deltaproteobacteria. Intrestingly, the Delataproteobacteria nitirite oxidizing bacteria familiy, Nitrospinaceae, are commonly found in the deep waters at P1200; however, no bacterial ammonia oxidizer was found in the database. This suggests tha ammonia oxidation is probably carried out by Archaea.

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307B The biodegradation of para-nitrophenol by river water biofilms: a functional marker approach Agnieszka Kowalczyk*1, Roger van Egmond2, Chris Finnegan2, Oliver Price2, Gary Bending1, Hendrik Schafer1 1The University of Warwick, United Kingdom, 2Safety & Environmental Assurance Centre, Unilever, United Kingdom

Standardized OECD biodegradation tests are conducted in the dark with a non-standardized inoculum from a range of environmental compartments. We investigated the effect of light on the biodegradation of para-nitrophenol (PNP) and the associated impacts on bacterial community composition. The biodegradation of PNP was followed over 100 days in water collected from the River Dene (Wellesbourne, UK). Total degradation of PNP at the concentration of 2mg L-1 occurred within 7 days in the dark, while degradation under light conditions was inhibited with only one replicate degrading (93%) after 56 days. Terminal Restriction Fragment Length Polymorphism of the 16S rRNA gene indicated that bacterial community structure was differentially affected by dark and light conditions and that isolate Pseudomonas syringae AKDH2 proliferated in the dark bottles but was absent from the light bottles. Characterisation of communities responsible for PNP degradation was based on a functional marker approach. Two PNP degradative genes were targeted: pnpA which is involved in the initial catabolism of PNP and mar, a lower PNP degradation pathway gene, also present in the degradation of many other aromatic pollutants. Functional diversity and gene abundance of pnpA and mar were assessed using microbial ecology techniques.

308B Effects of commercially available enzymes and antimicrobial peptides on bacteria communities in sydney harbour, Australia Agnete Krabbe Katholm*1, Henrik T. Jespersen2, Annemette Palmqvist3, Kjeld Schaumburg2, Eva Wallström4, Benni W. Hansen3 1Roskilde University, Denmark, 2Roskilde University, CISMI/FiBaC, Denmark, 3Roskilde University, Department of Environmental, Social and Spatial Change, Denmark, 4EnPro ApS, Denmark

All natural and artificial materials submerged in seawater experience a series of discrete physical, chemical, and biological events which results in the formation of a complex layer of attached organisms known as biofouling. Recently, the search for effective antifouling formulations with low adverse effects on non-target organisms has been focused on isolating marine natural compounds. However, the isolation, chemical synthesis or large-scale recombinant production of such natural biogenic agents is very costly. Another approach is to utilize biodegradable chemicals already isolated or formulated and industrially produced in large-scale. In this study commercially available enzymes and antimicrobial peptides were tested for their effects against bacterial attachment. Initially three enzymes and three peptides were immobilised by individual encapsulation in silica-based aerogel matrices. The aerogels were pulverised and dispersed individually and in combinations in self polishing hull paint which were applied on PVC plates and submerged into Sydney Harbour. Swaps were taken from the plates after one week and five weeks. Bacterial attachment and community composition from the different treatments were compared using automated ribosomal intergenic spacer analysis (ARISA). Results from the analysis will be presented and discussed.

309B Phylogenetic diversity of planktonic bacterial communities of Lake Hévíz Gergely Krett*, Katalin Jáger, Judit Makk, Károly Márialigeti, Andrea K. Borsodi Eötvös Loránd University; Department of Microbiology, Hungary

Lake Hévíz is the largest warm water natural spa lake of Europe. The curative effect of the water can be originated in the mineral salts derived from crater sprigs and the special sediment that forms a transition between moorish and volcanic types. Containing a combination of components with different origin, the sediment and the water of Lake Hévíz harbors peculiar bacterial communities, which may play important roles in the preservation of the natural state and the curative effect of the lake. Therefore, the aim of the present study was to gain comprehensive information about spatial and temporal distribution of planktonic microbiota. The phylogenetic diversity was studied by cultivation independent molecular biological Denaturing Gradient Gel Electrophoresis (DGGE) and cloning methods. Depth distribution of bacteria in the lake was studied following a sampling in November 2009, while horizontal and seasonal distribution in April, July and October 2010 and 2011 by DGGE

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based on 16S rRNA gene of Bacteria and Cyanobacteria specific primers. From a composite sample taken in April and October 2011, strain collections by using different culture media and 16S rRNA gene based clone libraries were established. Comparing the bacterial communities seasonal alternations in microbial community structures were observed according to the band numbers and intensities. Dominant DGGE bands were identified as representatives of class alfa-, beta- and gamma- Proteobacteria and phylum Actinobacteria. Clone library constructed was also dominated by members of class alfa- and beta- Proteobacteria.

310B Monitoring microbial species and total elemental of urban air particulate in Istanbul Mert Kumru*1, Melih Özgür Çelik2, Kerem Uzel3, Aslı Baysal1, Süleyman Akman1, Alper Tunga Akarsubaşı1 1Istanbul Technical University, Turkey, 2Charité - Universitätsmedizin Berlin, Germany, 3Bogazici University, Turkey

Istanbul is one of the most populated cities in the world residing over 13 million habitants. Since urban areas are major cause of air pollution, Istanbul is a major target for diverse chemical and physical substances that can cause mutagenicity. Airborne particulate matter (APM) is produced by human and natural activities. While people generate APM as a result of mass traffic, industrial activities and combustion processes; natural sources for APM are windblown dust and saline particles from sea. In addition to chemical and physical substances in the air; viruses, bacteria and fungi termed airborne microbial particles (MP), are introduced to atmosphere from agricultural, industrial and urban activities. Increase in the content of both APM and microbial particles MP are associated to increased cardiorespiratory diseases.

In this study, airborne bacteria were examined from outdoor sampling stations located on the urban (trafic related) zones in Istanbul. Total elemental airborne particulate matter is also surveyed in these samples and searched for significant correlations between APM and microbial particles. Air samples were collected onto a teflon filter by High Volume Air Sampling PM 2.5. DNA were extracted from the filters and later used for sequence analysis. Bacterial concentrations in the densely urban and highly trafficked areas were significantly lower than the rural greener areas. As a result, Micrococcus spp., and Staphylococcus spp., were dominant genera and amounted to over 90% of the total airborne bacteria. Corynebacterium spp., Bacillus spp., Enterococcus spp., and Streptococcus spp are also identified in the sampling locations with a relatively less frequency. Air particulate samples were digested by microwave assisted digestion system and after the digestion, the samples were analyzed by High Resolution Continuum Source Atomic Absorption Spectrometer. The results are compared after the microbial and total elemental analysis.

311B Microbial community dynamics of İSTAÇ landfill leachate wastewater treatment plant in İstanbul, Turkey Halil Kurt1, Aslıhan Demircan2, Mert Kumru1, Alper Tunga Akarsubaşı*1 1Istanbul Technical University Molecular Biology, Biotechnology & Genetics Research Centre, Turkey 2Yildiz Technical University, Faculty of Chemical and Metallurgical Engineering, Bioengineering Department, Turkey

Leachate is liquid material that drains from land or stocked material and the consistence depends on waste composition, waste age, and landfilling technology. Landfill leachate can be characterized as water based solution comprised four groups of pollutants: dissolved organic matter, inorganic macro components, heavy metals and xenobiotic organic compounds. İSTAÇ is one of the typical leachate wastewater treatment plant (WWTP) system with high chemical oxygen demand (COD) and ammonia.

The aim of this project is to analyze the microbial community dynamics of a domestic leachate treatment plant at different sampling times located in Istanbul Kemerburgaz (İSTAÇ AŞ) and to check correlation of temporal changes in microbial community versus performance parameters, such as ammonia and nitrate content, of the treatment plant.

Seven different samples were collected monthly from six different sampling points of İSTAÇWWTP. Population changes were monitored by 16s rDNA gene based PCR-DGGE methodology. DGGE patterns were analysed with BioNumerics software 5.1 (Applied Maths, 2008). Similarity matrix and

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dendrogram of the DGGE profiles were generated based on the Dice correlation coefficient (band-based) and unweighted pair-group method arithmetic average (UPGMA).

Bacterial communities were successfully amplified from all of the sampling points tobe analysed and community profiles were obtained. Differences in microbialcommunity profiles were correlated with variations in conventional parameters, especially with ammonia content, of the treatment samples. Occurrence of Archaeal amoA gene was not detected in any samples. On the other hand, ammonia oxidizing bacteria (AOB) occurrence was observed in nitrification pools and exit valves, as expected. However, AOB microbial community data was not sufficient for correlation with the conventional parameters.

Phylogenetic analysis shows that activated sludge samples of same seasonal time series were clustered closely together. The sample series that clustered on distinct branch of the dendogram was shown to have the lowest NH4-N and nitrate values. Further phylogenetic analysis of the samples may provide better understanding for correlation of conventional parameters and microbial community.

312B Distribution of ammonia-oxidizing archaea and bacteria along an ammonia gradient in oceanic sediment Lorenzo Lagostina*1, Lars Schreiber1, Mark Lever1, Thomas Evans2, Tobias Goldhammer3, Andreas Schramm4, Dorthe Groth Petersen1 1Center for Geomicrobiology, Department of Bioscience, Aarhus University, Denmark, 2Department of Geoscience, University of Bremen, Germany, 3Center for Marine Environmental Sciences, University of Bremen, Germany, 4Department of Bioscience, Microbiology, Aarhus University, Denmark

The nitrogen cycle in marine sediments is complex and still not completely elucidated. The recent discovery of ammonia-oxidizing archaea (AOA), and recent findings that these archaea are widespread and in many environments outnumber ammonia-oxidizing bacteria (AOB), indicate that AOA play a significant role in the nitrogen cycle. Yet, niche partitioning between AOA and AOB is poorly understood, although ammonia concentrations have been proposed as a key factor. The aim of this study was to examine the role of ammonia for the distribution, diversity, and abundance of AOA and AOB in oceanic surface sediment. Sediment was retrieved along a transect between the coast of Namibia and the South Atlantic Gyre at the RV Meteor cruise M76 in spring 2008. The transect covered 7 stations from which we have geochemical profiles (NH4

+, TOC, SO42-, alkalinity) from

surface to 100 cm below the surface (cmbsf). Three representative stations based on high (H), medium (M) and low (L) ammonia concentrations (5 cmbsf) were chosen in order to analyse detailed molecular data from three depths (0-1, 6-10 and 12-16 cmbsf). Community DNA was extracted by two different DNA extraction methods to yield the most comprehensive analyses allowing us to analyses biases in our results introduced by DNA extraction protocols. The abundances of AOA and AOB were determined by qPCR targeting of archaeal and bacterial amoA genes, which encode ammonia monooxygenase, the key enzyme for ammonia oxidation. The diversity of surface sediments was analysed by cloning and sequencing bacterial and archaeal amoA genes, and 16S rRNA genes of the betaproteobacterial AOB. Preliminary results showed that AOB outnumbered AOA in the top sediment independently of ammonia concentrations with AOB/AOA ratios ranging from 11 to 112. The relative abundance of AOA to AOB increased with depth across the 3 sites (AOA/AOB ratio from 1 to 21). Across all sites, AOA communities were more diverse and had higher richness compared to AOB communities, but in general the diversity increased for both AOA and AOB when moving into low ammonia concentration stations. Based on phylogenetic trees, we found no clear distribution of AOA species between the three sites; nevertheless, there were indications of distinct ecotypes and distributions among the AOB, which all belonged to the genus Nitrosospira.

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313B Archaeal and bacterial communities in the potential gas hydrate bearing sediments at offshore SW Taiwan Mei-Chin Lai*1, Chuan-Chuan Hung1, Shu-Yao Wu1, Shu-Jung Lai1, Jiun-Yan Ding1, Saulwood Lin2, Tsanyao Frank Yang3, Po-Chun Chen4 1National Chung Hsing University/Department of Life Sciences, Taiwan, 2National Taiwan Univ/Institute of Oceanography, Taiwan, 3National Taiwan Univ/Dept. of Geosciences, Taiwan, 4Central Geological Survey, Taiwan

The deep sub-seafloor biosphere is among the least-understood habitats on Earth, even though the huge microbial biomass plays a potentially important role in long-term controls of global biogeochemical cycles. Due to the interest in marine gas hydrate, the research team from Taiwan, supported by the Central Geological Survey (CGS), has been demonstrated the intensive Bottom Simulating Reflectors distribution, high methane and dissolved sulfide concentration, high sulfate reduction rate, shallow depth of sulfate-methane-interface and the carbon isotopic composition of the authigenic carbonates at SW offshore Taiwan that indicated this area are potential gas hydrate region.

In this study, the composition of the Archaeal and Bacterial communities were determined by 16S rRNA phylogenetic analyses of clone libraries derived from DNA extracted from sediment samples obtained by piston cores from Marion Dufresne 178 cruise. The phylotype analysis of all cores represented similar phylotype groups as reported in cold seep, hot vent and gas hydrate habitats undersea. The microbial composition of core sediments from both passive and active continental margin represented archaeal phylotypes of Marine Benthic Group D, ANME, SAGMEG, DSAG and MCG and bacterial phylotypes of Planctomycetes, Chloroflexi, candidate JS1 division and Proteobacteria. Among them, the total clones of bacterial phylotypes Chloroflexi, Planctomycetes, JS1 and archaeal phylotype DSAG are dominant which represented the prokaryotic community structure of the gas hydrate bearing sediments. In addition, gas hydrate related ANME2a was detected in these cores and showed 99% similarity with ANME2a obtained from the above of gas hydrate at Hydrate Ridge. The characterization of microbial community at gas hydrate area is essential to gain a better understanding of microbial diversity, contribution and the effects of microbial activities in hydrate ecosystems.

314B Assessing biodiversity of thermophilic fungal species in composts using conventional and molecular techniques Adrian Langarica Fuentes*, Geoff D. Robson, Pauline S. Handley University of Manchester, United Kingdom

Thermophilic fungi are small group of organisms that have a minimum temperature for growth at or above 20˚C and a maximum above 50˚C. From a biotechnological perspective, thermophilic fungi are of great interest due to their important role in the composting process and their ability to degrade recalcitrant materials. In this study, the biodiversity of thermophilic fungi in two different commercial composts was investigated using traditional culture-based methods and two culture-independent techniques: denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP). Compost A was produced using forestry cuttings, while compost B used garden and municipal solid waste. Thermophilic fungi were isolated and identified from compost A (6 species) and B (7 species), respectively, using culture-based techniques. However, DGGE and T-RFLP analysis of these composts revealed a more complex fungal diversity. DGGE confirmed the presence of some thermophilic organisms that were not recovered using conventional methods and also revealed the presence of several unculturable organisms that may have a major role in the community. T-RFLP was the most sensitive technique to monitor the dynamics of the fungal community while providing valuable diversity indicators. Statistical analysis of DGGE and TRLFP data showed good correlation between the two techniques. Comparing DGGE and T-RFLP with isolates recoverable by conventional culturing has shown that culture-based methods, which have been predominantly used previously to investigate the fungal population of composting environments, are insufficient and misleading.

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315B Isolation and characterization of Cr(VI)-reducing bacteria from a chromium contaminated aquifer in Guanajuato, México with potential application in bioremediation Paloma Lara*, Enrique Morett, Katy Juárez Universidad Nacional Autónoma de México/ Instituto de Biotecnología, Mexico

Biotransformation of Cr(VI) to less-toxic Cr(III) by chromate-resistant and reducing bacteria has offered an ecological and economical option for chromate detoxification and bioremediation. Chromium reducing microorganisms with high levels of resistance have taken great interest in recent years due to their potential application in bioremediation processes because it is a safe and cost-effective technology alternative to the expensive traditional physical-chemical methods. The present study was aimed to the isolation of indigenous Cr(VI)-resistant and Cr(VI)-reducing bacteria from an aquifer located in Guanajuato, Mexico, contaminated over a long period of time by chromate. First, we isolated some strains from anaerobic enrichment cultures, which presented resistance of high levels of Cr(VI) and also were capable to reduce it to Cr(III). The isolated colonies presented diverse morphologies. The analysis of 16S rDNA allowed us to identify the genera and species of some isolates, showing a great phylogenetic diversity. Fifteen different facultative anaerobe microorganisms belonging to the phylum proteobacteria, firmicutes and actinobacteria showed high levels of resistance to chromium with a minimum inhibitory concentration of about 1195 mg/l de Cr(VI) in aerobiosis. Some isolates were related to bacteria with capabilities to reduce metals, for example Streptomyces sp., Bacillus sp. and Antrobacterium sp. One of them is closely related to Klebsiella pneumonie KCTC 2242, it was assayed for Cr(VI) and Fe(III) reduction, using fermentable substrates such as citrate, and it forms a hydrogel consisting of capsular polysaccharides with high affinity to metals as a resistance strategy. This approach permitted selection of some bacterial strains, which could be used for bioremediation of Cr(VI)-polluted environments, demonstrating the utility of using a selection strategy for indigenous Cr(VI)-reducing bacteria in a biostimulation process.

316B Paenibacillus hankukensis sp. nov., isolated from soil Geon-Hyoung Lee1, Sung-Jun Cho*1, Sung-Heun Cho2, Suhk-Hwan Park1, Seung Bum Kim2 1Kunsan National University/Dept. of Biology, South Korea, 2Dept. of Microbiology & Molecular Biology, Chungnam National University, South Korea

Gram-negative and strictly aerobic rod-shaped bacterium, designated DS80T, was isolated from an island soil. Strain DS80T grew optimally when cultured at temperatures between 15 and 40℃, and at pH values ranging from 5.0 ~ 9.0. A phylogenetic analysis based on the comparisons of the 16S rRNA gene sequences showed that the isolate was affiliated with a cluster of strains within the genus Paenibacillus. The level of 16S rRNA gene sequence similarity between DS80 and Paenibacillus assamensis GPTSA11T was 96%. The G+C content of the genomic DNA was 44.0 mol%, and the major fatty acids of the strain were anteiso-C15:0 (41.38 %), iso-C15:0 (12.42 %), iso-C16:10 (8.74 %), and C16:1ω11c (6.148 %). The strain contained MK-7 as the major menaquinone. The major polar lipids were phosphatidylinositol, phosphatidylglycerol, phosphatidylethnolamine and diphosphatidylglycerol. Therefore, on the basis of phenotypic and phylogenetic data, strain DS80T should be recognized as a new species of the genus Paenibacillus, for which the name Paenibacillus hankukensis sp. nov. is proposed (type strain = DS80T = JCM 17278T = KCTC 13833T).

317B Phylogenetic Characterization of Actinobacteria in a Bamboo (Sasa boreali) Forest Soil by Using Pyrosequencing Hyojin Lee*, Kyungsook Whang Department of Microbial & Nano Materials, Mokwon University, South Korea

Our previous paper identified bacterial populations in dominants bamboo (Phyllostachys bambusoides, Phyllostachys nigro var. henonis, Sasa boreali and Phyllostachys nigra f. punctata) forest soil at Damyang in Korea. A quantitative evaluation of microbial populations in a various bamboo forest soil was examined. The numbers of actinobacteria in each different layers of bamboo forest soil ranged from 2.7×106 to 2.7×108 CFU/g. Sasa boreali bamboo forest soil was detected with relatively higher actinobacteria. Ninety-seven percent of the total actinobacterial isolates were belonged to the genus Streptomyces, suggesting the dominance of actinobacteria. These actinobacterial isolates were categorized as four groups. This investigation evaluates the characterization of bacterial populations in a Sasa boreali bamboo forest soil by using V2-V3 16S rRNA gene pyrosequencing. The samples were

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carefully divided into litter and rhizoshpere soil. The number of classified sequences per sample ranged from 4,400 to 5,328. Bacterial diversity was more phylum rich in rhizoshpere soil than litter in Sasa boreali forest soil. The dominant taxonomic groups across all samples (> 4% of all sequences) were Proteobacteria, Bacteroidetes, Actinobacteria and Acidobacteria. Especially, Actinobacteria community composition and diversity showed significant differences between litter and rhizoshpere soil. Fourteen percent of the total sequences from bamboo litter was Actinobacteria, they were categorized in five family groups (> 8%); Pseudonocardiaceae, Microbacteriaceae, Streptomycetaceae, Nocardioidaceae and Micromonosporaceae. Four percent of the total sequences from rhizoshpere soil were actinobacteria which assigned to two dominant family groups; Gaiellaceae and Streptomycetaceae. Among these families, Streptomycetaceae was the predominant taxon and constituted 8.1-13% of total actinobacteria sequences. We determined their phylogenetic profiles, 110 sequences belong to Streptomycetaceae from litter were classified into 23 subgroups of Streptomyces. Eleven sequences from rhizosphere soil were classified into 5 subgroups of Streptomyces.

318B Rhizosphere bacterial communities of bacterial wilt resistant or susceptible tomato cultivars and a non-host baechu cultivar Hyoung Ju Lee*1, Byung Kwon Kim2, Myung Hwan Lee1, Hyun Gi Kong1, Eun Joo Jung1, Hae Jin Joo1, Jihyun F. Kim2, Seon-Woo Lee1 1Dong-A University, South Korea, 2Yonsei University, South Korea

Bacterial wilt caused by Ralstonia solanacearum is a soil-borne disease causing lethal wilt on many Solanaceae plants. Although several tomato lines resistant to bacterial wilt have been reported, the nature of host resistance to R. solanacearum in tomato plants has been uncharacterized. Here, we investigated the bacterial community structures of rhizosphere soils from different plants, such as resistance tomato cultivar Hawaii 7996 and susceptible tomato cultivar Moneymaker and a non-host baechu (Korean cabbage). We analyzed microbial communities from rhizosphere soils using highly paralleled pyrosequencing approach of 16S rRNA gene. Rhizosphere soils were obtained from plant cultivars grown in a green house at active growth with initial flowering (AG) stage of tomato plants and at fruiting with senescence (FS) stages of tomato plants. Corresponding bulk soils without any plant cultivation were obtained at the same time. Bacterial 16S rRNA genes were amplified using barcoded primers targeting the V1-V3 region of 16S rRNA and microbial 16S rRNA genes were determined using 454-pyrosequencing of the amplicons. The 16S rRNA gene sequences were analyzed using by Ribosomal Database Project Pyrosequencing Pipeline and MOTHUR. Rhizosphere soils of both AG stage and FS stage represented the lower species richness and evenness compared with bulk soil control. Principal coordinate analyses revealed that bacterial communities in plant rhizosphere were different from bulk soil and they were also different each other among plant cultivars Moneymaker, Hawaii 7996 and baechu, especially at AG stage. At FS stage with lower temperature, species richness and evenness increased in rhizosphere compared to those of AG stage. Difference in bacterial communities was the higher at AG stage among plant cultivars than that at FS stage. Members of the Proteobacteria and Bacteroidetes dominated in rhizosphere at both growth stages, while members of Bacteroidetes were found at lower abundance in bulk soil. Our result suggested that plant rhizosphere-associated bacteria increase when plants were actively growing and tend to decrease at later stage of plant growth. Difference in bacterial communities in rhizosphere was apparent among plant cultivars.

319B The malodor deodorizing effect of Effective Microorganism (EM) in food garbage Young Ok Lee*, Hong-Jae Choi Daegu University, South Korea

In order to monitor the malodor deodorizing effect depending on the presence/absence of Effective Microorganism (EM) developed by Japanese Higa, the bacterial community structure of 2 commercial EMs (A/B) and those cultured EMs in a Korean food garbage treatment plant was analyzed by pyrosequencing method, and those 10 malodor compounds(6 aldehydes, 3 sulfuric compounds, NH3) were determined using High Performance Liquid Chromatography(HPLC), Gas Chromatography(GC), spectrophotometer respectively. For pyrosequencing of EM with Roche 454, those DNA was extracted using Qiagen DNeasy Kit and amplified using PCR with 27F/518R primer.

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As result, the bacterial species composition between 2 commercial EMs (A/B) and those cultured EMs in the treatment plant was similar: the bacteria existing in common were Lactobacillus sp. and Acetobacter sp. But Acetobacter sp. (76%) was dominated in the commercial and cultured EM-A used for deodorizing the food garbage in spring. when the concentration of various aldehydes malodor was reduced. But the commercial and cultured EM-B used in summer comprized different Lactobacillus sp. (93%). And at that time aldehyde compounds increased, but decreased S-compounds a little bit. That is, function of EM of 2 companies was different. So in Fall, the mixed EM of 2 companies in ratio of 7:3(A:B) was used and reduced all of 10 kinds of determined malodors. However, even if EM was added to the food garbage in hot summer (30℃), the malodor could not be controlled efficiently; some kind of malodor compounds increased up to 60 times compared to other seasons due to the increased microbial activity according to temperature.

In conclusion, temperature affects mainly the concentration of malodor compounds generated from the food garbage. So when the temperature was under around 20℃, the malodor could be controlled by EM composed of Lactobacillus sp. and Acetobacter sp. However, when the temperature was higher than 20℃, it is most likely to exceed the emission standard of Korean malodor control law.

320B Microbial ecology surrounding methylated sulfur and selenium compound formation in freshwater Celine Lesaulnier*1, Gabriel Singer1, Jutta Niggemann2, Cédric Gerard3, Xavier LeCoz3, Claus Pelikan1, Thorsten Dittmar2, Alexander Loy1 1University of Vienna, Austria, 2Max Planck Institute, Germany, 3Nestlé Research Center, Switzerland

The sources and mechanisms of dimethylsulfide (DMS) and dimethylselenide (DMSe) formation are of significant interest given their importance in global sulphur and selenium cycling. Their discovery in freshwater systems has led to a growing interest in understanding these processes in these environments. However, the microorganisms responsible, the mechanisms they utilize, and conditions favouring DMS and DMSe formation remain poorly understood. Here we examined spring water microbiota as a low-diversity model system to study these processes.

Microbial community time-series analyses were performed on two different spring waters, one of which is prone to the formation of DMS and DMSe. Growth profiles were quantitatively determined between 1 and 56 days post storage in polyethylene terephthalate (PET) containers via epifluorescence microscopy in combination with DAPI (4',6'-diamidino-2-phenylindole) DNA staining. For each sample, total of 120 chemical parameters were measured to monitor changes in water chemistry throughout the sampling period, and bacterial community composition (planktonic and biofilm) were simultaneously determined for each sample using 454 pyrosequencing and Sanger sequencing of 16S rRNA gene amplicons.

Greater species richness was noted in both waters at earlier time-points, although environmental conditions rapidly resulted in the dominance of Betaproteobacteria. We found characteristic microbial communities for each water with little overlap between them. We further observed differences between biofilm and planktonic microbiota of the same water. Results were further confirmed by fluorescence in situ hybridization (FISH). Results from the time-series were then compared to 5 subsamples of the DMS/DMSe-prone water which presented clear formation of these compounds, as predetermined by chemical analyses. Microbial community composition differed significantly between samples and showed greater microbial richness than non-DMS/DMSe waters. We further identified putative candidate organisms for DMS and/or DMSe formation.

The mechanisms of DMS and DMSe formation were investigated by adding various chemical precursors and inhibitors, at 2 different concentrations, to the DMS/DMSe-prone water within 24 hours of incubation. Samples were monitored over a four week period and conditions which generated DMS/DMSe formation were determined. These results suggest the most plausible metabolic pathways for their formation in this environment.

As microbial growth is naturally mediated by the availability of dissolved organic carbon (DOC), we performed Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to determine the DOC composition in these two waters. By combining two high resolution techniques (454

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sequencing and FT-ICR-MS) we aim to link DOC and its turnover, to microbiota dynamics and thereby understand the formation of volatile sulphur and selenium compounds in these environments.

321B Abundance and diversity of Sphingomonas in Shenfu petroleum-wastewater irrigation zone, China Hui Li*, Lisha Zhou, Ying Zhang, Siqin Han, Hui Xu Institute of Applied Ecology, Chinese Academy of Science, China

The genus Sphingomonas was proposed in 1990 and had received increasing attentions due to its ubiquity and metabolic versatilities, especially its ability for utilizing polycyclic aromatic hydrocarbons (PAHs) as the sole source of carbon and energy. However, investigations into the richness, diversity and population structure of Sphingomonas in polluted ecological niches have not been well documented due to lack of suitable non-degenerate genus-specific primers. In the present study, a new primer set targeting the Sphingomonas 16S rRNA gene was designed, and the specificity was tested with four petroleum-contaminated soils by construction of clone libraries and further restriction fragment length polymorphism analysis. Subsequently, real time quantitative PCR and denaturing gradient gel electrophoresis (DGGE) assays were used to evaluate the abundance and diversity of Sphingomonas in the Shenfu irrigation zone, the largest petroleum-wastewater irrigation zone in China with 70-year exposure to petroleum contaminants. Fifteen sampling sites different in their relative geographical position, irrigation history, and soil management were selected from up- to downstream.

Results showed that the newly developed genus-specific primer set SA/429f-933r was more suitable for specific detection of Sphingomonas members in soils than previous reported primers. Ninety percent of the sequences retrieved from soil clone libraries by this specific primer set were related to Sphingomonas. Results from real time PCR showed a widespread distribution of Sphingomonas in Shenfu irrigation zone, with 1.54×107 to 8.34×108 gene copies per gram of soil. Significant correlation (p<0.05) was observed between the Sphingomonas abundance and the ratios of PAHs to total petroleum hydrocarbon (TPH). The fraction of Sphingomonas to total bacteria in soils changed greatly in response to different contamination level and cultivation management type, and the proportion was ranged from 0.77 to 40.22%. The relative Sphingomonas abundance in upland soils was higher than that in paddy soils. DGGE profiles revealed that Sphingomonas population structures differed greatly in different sites. Cluster analysis of DGGE banding patterns indicated that the population structure of Sphingomonas was correlated with the management type and the geographical location. The Sphingomonas diversity was not statistically (p>0.05) correlated with the contamination level. Phylogenetic analysis of the re-amplified DGGE bands revealed that the dominant Sphingomonas in Shenfu irrigation zone may represent the new species.

In addition, 7 cultured Sphingomonas isolates were screened from contaminated soil by using genus-specific primers combined with streptomycin resistant plate. Sphingomonas 4c was the most promising strain for biodegradation of PAHs contaminated soils. The degradation rate for fluorene and phenanthrene by Sphingomonas 4c was 87.2% and 64.8%, respectively.

We can conclude that the newly designed Sphingomonas-selective primers could be used for specific and direct detection of as much as known Sphingomonas in petroleum-contaminated soils. The significant correlation between the abundance and the ratios of PAHs to TPH suggested an important role of Sphingomonas in PAH bioremediation.

322B Identification of novel bacterial copper membrane monooxygenases (CuMMO) related to C1-C4 alkane oxidation in a natural hydrocarbon plume Meng Li*, Sunit Jain, Brett Baker, Chris Taylor, Gregory Dick University of Michigan, USA

The copper membrane-bound monooxygenase (CuMMO) family includes particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO), which play key roles in global carbon and nitrogen cycling. In addition to catalyzing oxidation of methane and ammonia, CuMMOs are also proposed to oxidize the C2-C4 alkanes with alternate functions. To extend our understanding of the distribution and activity of the CuMMOs with diverse functions in natural environments, we analyzed the metagenomes and metatranscriptomes of hydrocarbon-rich Guaymas Basin (GB) hydrothermal

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plumes and nearby background waters in the Gulf of California for the presence and expression of genes encoding CuMMOs.

High-throughput random shotgun sequencing of community DNA and RNA from hydrothermal plume and background samples was conducted with 454 GS FLX Titanium and Illumina HiSeq2000. De novo assembly was used to reconstruct partial genomes and transcriptomes.

At least six distinct phylogenetic groups of bacterial CuMMO are present and transcriptionally active in both hydrothermal plume and background waters: Methylococcaceae-like ethane-oxidizing bacteria, Actinomycetales-like C2-C4 alkane oxidizing bacteria, three clades of type I methanotrophs (a symbiont-like group and two methane-oxidizing marine groups, OPU1 and OPU3), and a group of unknown affiliation/function. Corresponding 16S rRNA sequences of these groups were also recovered. Comparative analysis shows that the bacterial CuMMOs identified here have low sequence similarity with previously reported sequences, especially those groups related to C2-C4 alkanes oxidation. Although they share conserved amino acids that serve as ligands for copper centers with known CuMMOs, they also show changes at the variable metal site, indicating the coexistence of several divergent bacterial CuMMOs with potentially different functions in the GB hydrothermal system. At a finer phylogenetic level, two major phylotypes (OPU1 and OPU3) display frequent single-nucleotide polymorphisms in their operons, further highlighting the diversity of CuMMO coding genes in GB plumes. Genes encoding five bacterial CuMMO groups show dynamic transcript levels in plume and background samples.

We conclude that there is a remarkable diversity of novel bacterial CuMMOs related to C1 to C4 alkane oxidation that are simultaneously active in deep waters of the Gulf of California. These results imply a broader distribution of novel CuMMOs in the deep sea than previously recognized, and highlight gaps in understanding regarding the diversity and function of these enzymes in natural environments.

323B Identification of bacteria and T4-type bacteriophages that assimilate carbon from root cap cells under aerobic and anaerobic soil conditions using a DNA-SIP approach Yong Li*, Takeshi Watanabe, Jun Murase, Susumu Asakawa, Makoto Kimura Nagoya University, Japan

Plant roots foster microorganisms in their rhizosphere by supplying various organic substrates such as root exudates and excretions, mucilages, slough-off root cap cells and dead/moribund old root tissues. Root cap cells are substrates for rhizosphere microorganisms, representing 5-10% of the total carbon deposited. Although root cap cells are an important substrate for microorganisms inhabiting the rhizosphere, little attention has been paid to the microorganisms responsible for the decomposition of sloughed root cap cells because of the methodological difficulties to clearly separate responsible microorganisms from those grown by other organic substrates of root-origin in the rhizosphere. To elucidate the bacterial communities responding for the decomposition of sloughed root cap cells and whether T4-type phages infecting them, we prepared rice callus cells as a model material for root cap cells, and 13C-labelled and unlabelled callus cells were allowed to decompose in aerobic and anaerobic soil microcosms for 56 days. DNA was extracted periodically from the soils and subjected to polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) after density gradient ultra centrifugation. Comparison of DGGE band patterns of isopycnically fractionated samples from 12C- and 13C-callus treatments showed many bands in the heavy buoyant density fractions only for 13C-callus treatment in every sampling time. Sequencing of characteristic DGGE bands in the heavy fractions from 13C-callus treatment showed that α-Proteobacteria, γ-Proteobacteria, Sphingobacteria and Actinobacteria were dominant callus-C incorporators in aerobic soil incubations. In contrast, strictly anaerobic bacteria such as Clostridium, Geothrix and Holophaga were dominant decomposers of rice callus cells under anaerobic soil conditions. Many 13C-labelled g23 fragments belonging to Paddy Group V, VIII and IX were obtained only in aerobic soil incubation, which indicates that phages mediate the lysis of bacteria that assimilated the carbon from callus cells under the aerobic soil conditions. The present study indicates that specific bacterial species are involved in decomposition of riceplant root cap cells and they are different from each other under aerobic and anaerobic soil conditions. It also evidenced the carbon flow of root cap cells to the T4-type phage-mediated microbial loop under aerobic soil conditions.

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324B Bacterial and archaeal diversity in soil from Atlantic forest of Sao Paulo State Julia Elidia Lima*1, Armando Cavalcante Franco Dias2, Emiliana Manesco Romagnoli1, Ademir Durrer1, Fernando Dini Andreote1 1ESALQ - University of Sao Paulo, Brazil, 2CENA - University of Sao Paulo, Brazil

The Atlantic Forest biome consists of many ecosystems and is considered one of the world's biodiversity hotspots. It extends from Rio Grande do Sul to Piauí covering nearly 15% of the country. However, approximately 93% of the original Atlantic Forest has been devastated. Soils are often poor (low nutrient availability) in this biome, but exhibit high rates of organic matter decomposition, low nutrient loss by leaching, and high nutrient cycling. Therefore, this environment harbors complex and efficient microbial communities that participate in these processes, although their role is still poorly described. Thus, this study assessed several factors directly related to bacterial and archaeal community composition at 3 Atlantic Forest sites in an altitudinal gradient: Santa Virgínia, Picinguaba, and Restinga. Based on culture-independent analyses, PCR-DGGE profiles indicated that bacterial and archaeal community structures at these sites are more dependent on vegetation than soil physical and chemical attributes. Additionally, abundance of these communities, determined by real-time PCR, was similar at the 3 sampling sites, with 109 and 108 copies of 16S rDNA gene for Bacteria and Archaea, respectively. Regarding the taxonomic groups present at these sites, bacteria predominantly belonged to the phyla, Acidobacteria, Verrucomicrobia, and Proteobacteria (highlighting Acidobacteria, the dominant group with an average of 55.9%) and archaea were similar to Euryarchaeota and Crenarchaeota groups, highlighting Crenarchaeota with an average of 84%. Thus, this study took a novel approach to illustrate the detailed composition of Bacteria and Archaea in Atlantic Forest soil, pointing out alterations in these communities due to different characteristics specific to each sampling site, mainly driven by vegetative floristic composition.

325B Microarray and 15N isotope analyses of denitrifying communities in New River Estuary, USA Jessica Lisa*1, Amal Jayakumar2, Craig Tobias3, Bongkeun Song1, Bess Ward2 1UNC Wilmington, USA, 2Princeton University, USA, 3University of Connecticut, USA

Denitrification is a major microbial pathway for nitrate removal in sediments in estuarine ecosystems. Sediment denitrifying assemblages are very diverse, but, the relationship between community structure and activities of denitrifying bacteria is not fully understood in eutrophic estuaries. In order to reveal the differences in denitrifying community structures along estuarine gradients, we conducted nirS gene microarray analysis with sediment samples collected at four sites in the New River Estuary, NC, USA. Denitrification activities were measured with 15N tracer incubation experiments using Isotope Ratio Mass Spectrometry. A customized microarray containing 165 nirS gene probes (archetypes) was designed from all available nirS sequences to represent sequences separated by >15% divergence. Abundant archetypes were defined as those contributing >1% to the total fluorescence signal. The 68 abundant archetypes represent nirS sequences previously found in other estuarine, wetland, groundwater and water column environments. Spatial variation of dominant archetypes was observed along the New River Estuary. Canonical correspondence analysis was conducted to determine the environmental parameters influencing relative abundance of the nirS archetypes. Salinity was positively correlated with the nirS archetype of Brachymonas denitrificans, while two probes designed from sequences detected in Chesapeake Bay and Washington Coastal sediments were positively associated with increasing sulfide concentration. Potential denitrification rates were positively correlated with percent organics, sulfide, nitrate, ferrous iron and ammonium. Interestingly, two dominant denitrifying archetypes obtained from Arabian Sea water column and Chesapeake Bay sediment sequences were positively correlated with potential denitrification rates. These findings suggest particular geochemical features in the New River Estuary influence the distribution of the denitrifying microbial community, which in turn may affect the nitrate removal capacity of the estuary. Further analysis identifying geochemical parameters associated with nirS diversity and denitrifying activity will be conducted in order to reveal the linkage between structure and function of denitrifying communities in this eutrophic estuary.

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326B Polyphasic approach for assessing changes in marine microbial community under the double stress of UV and diesel oil Shiu-Mei Liu*, Chi-Yuen Yen, Hung-Hsuan Chien Institute of Marine Biology, National Taiwan Ocean University, Taiwan

Most crude oil-contaminated shore-lines were affected by the tidal actions. At the low tide, surface of these ecosystems are usually low in sea water and exposed to strong UV radiation in subtropical or tropical coastal areas. Thus, UV resistant hydrocarbon-degrading microorganisms probably are better players in cleaning up surface of these contaminated sites.

In this study a multidisciplinary strategy e.g. traditional techniques such as enrichment and isolation of UV resistant diesel oil degrading microorganisms from the contaminated sea water and sea sand, and molecular biological techniques such as denaturing gradient gel electrophoresis (DGGE) and pyrosequencing were used to investigate the change of microbial community structure in the sea ater and sea sand during enrichment of hydrocarbon degrading microorganisms after UVA and B radiation.

21 yeast strains and 1 bacterial strain were isolated after UV A and B radiation and diesel oil enrichment. Bacterial strain Ochrobactrum tritici showed the strongest total petroleum hydrocarbon (TPH) degrading ability (removed 48.9% of 1% crude oil during 30 days incubation) after UV A and B radiation. Among the isolated yeast strains, 6 strains of Candida tropicalis showed stronger TPH degrading ability (remove 33.1-40.8% of crude oil during 30 days incubation) after UV A and B radiation. Oil degrading ability of these yeast strains were correlated with their membrane hydrophobicity. Bacterial strain (remove 76%) and yeast strains (remove 45.7-67.1%) all showed stronger oil degrading ability without UV A and B radiation. Candida tropicalis strains posses lipase and catechol 1,2-dioxygenase, but not catechol 2,3-dioxygenase activity. 89% of the bacterial cells and 92-99% of Candida cells survived after under UVA and UVB radiation for 5 h. E coli was not survived under this conditions. Under UV C radiation (800 J/M2), all the Candida tropicalis strains had similar ability for UV resistance as that of Deinococcus radiodurans, however, bacterial strain could not survived under this conditions.

DGGE patterns and prosequencing data also showed the number of bacteria decreased following UV A and B radiation, and hydrocarbon enrichment.

327B The effect of whey permeate additions on growth performance, gastrointestinal morphology, and colonic microbiota of postweaned piglets Sarmauli Manurung*1, Tim Kare Jensen1, Bent Borg Jensen2, Lars Mølbak3 1Technical University of Denmark, National Veterinary Institute, Denmark, 2University of Århus, Department of Animal health, Welfare, and Nutrition, Denmark, 3Chr Hansen A/S, Animal Health and Nutrition, Denmark

The abundant of lactose as simple carbohydrate in whey permeate was hypothesized to improve gastrointestinal health of piglets. On the other hand, it was reported that lactose consumptions at high levels may increase the risk of osmotic diarrhea. In this study, one hundred piglets (weaned at 28 d, 7.7 kg live weight) were involved in experiments to investigate the effect of adding different concentrations of a commercial whey permeate (Variolac®) product on clinical symptoms, intestinal morphology, weight gain, and colonic microbiota communities. The piglets were fed basal diet with different levels of in-feed whey permeate for 35 days: no Variolac®, 60 g/kg Variolac®, 120 g/kg Variolac®, 180 g/kg Variolac®, and 240 g/kg Variolac®. After the first 2 weeks, 50 piglets were sacrificed and tissue samples were taken from duodenum, jejunum, ileum, and colon for morphological measurements. Digesta samples were obtained from colon for determination of microbial communities. The analyses of microbial communites were performed by next generation sequencing (Illumina) on the 16S rRNA gene V1 region and the results were analysed using a k-mer oligo matching program (Simrank2). The remaining 50 piglets continued to receive diets until week 5. None of the experimental piglets showed adverse clinical symptoms throughout the study. There were no differences in the total length of villous height and crypt depth in ileum or colon, respectively. There were no differences in the weight gain of piglets neither after the first 2 weeks nor at the end of 5 weeks experiment. The core communities (99.9 % reads) in the colonic digesta included the following phyla: Firmicutes, Bacteriodes, Tenericutes, Protobacteria, Actinobacteria, TM7, Spriochaetes, Acidobacteria, and Chloroflexi. Addition of 60 g/kg Variolac® in the diet increased the amount of

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Firmicutes (P < 0.01) relative to control. At the highest inclusion level (240 g/kg), the number of Bacteroides increased relative to the other treatment groups. In conclusion, addition of Variolac® as high as 240 g/kg into postweaned piglets was well tolerated as no maldigestion of carbohydrate was observed. The changes of colonic microbiota communities were observed only at extreme levels (lowest or highest additions).

328B Sulfur-cycling bacteria dominate marine harbour structures with past evidence of accelerated low water corrosion (ALWC) Florence Marty*1, Sandrine Païsse2, Jean-François Ghiglione3, José M. Sánchez-Amaya4, Hervé Gueuné5, Laurent Quillet2, Mark C.M. van Loosdrecht1, Gerard Muyzer1 1Delft University of Technology, Netherlands, 2Université de Rouen, UFR des Sciences/2M2C, UMR CNRS 6143, France, 3Université Pierre et Marie Curie (Paris 6)/ Laboratoire d'Océanographie Biologique de Banyuls, France, 4Universidad de Cádiz/ Laboratorio de Corrosión, Spain, 5CORRODYS, Centre de Corrosion Marine et Biologique, France

ALWC (Accelerated Low Water Corrosion), an aggressive and localized form of corrosion is of particular concern worldwide. Currently, the mechanisms and factors triggering its occurrence are still not fully understood. Previous culture-based studies evidenced themicrobiologically influenced nature of this corrosion process with the likely involvement of sulfate-reducing bacteria and sulfur-oxidizing bacteria. To gain a better knowledge of bacterial communities and functionalities associated with ALWC, corrosion deposits with typical aspects of accelerated (that is ALWC-suspected) and normal (that is NLWC) low water corrosion events were recovered from a 50-years old harbour steel structure with incidence of ALWC since the 80’s. The different layers composing the deposits were analysed with molecular methods. Comparative analysis of the total and the metabolically active bacterial communities by PCR-DGGE and PCR-CE-SSCP of 16S rDNA genes and transcripts indicated different community structure between inner layers, close to the steel surface and external layers, exposed to the seawater, irrespective of populations’ state (present or active) and sample type. Phylogenetic analysis of sequences obtained from a majority of DGGE bands and quantification of band intensities evidenced the dominance of active populations belonging to α- and δ-Proteobacteria and Bacteroidetes and lower proportion belonging to g-Proteobacteria, Firmicutes and cyanobacteria. A spatial segregation of a majority of SRB-affiliated and SOB-affiliated members, confined respectively to inner layers and outer layers, were particularly evidenced in ALWC-suspected samples correlated with mineralogical heterogeneity between layers. Also, these active populations were more dominant and/or diverse in ALWC-suspected deposits than in NLWC deposits.This tendency was further confirmed for SRB-related populations by qPCR (dsrB transcripts/16S rRNA) and by PCR-DGGE analysis of drsB transcripts and differences in relative ratios of SRB-related phylotypes could be observed. The bacterial diversity and mineralogical heterogeneity observed suggested key biomineralization processes. Furthermore, the presence of active hydrogenotroph and/or specialized SRB-related members known for example for their particular efficiency in catalysing iron oxidation (Desulfopila corrodens) or for example for their ability to disproportionate sulfur compounds (Desulfocapsa sp.) together with particular SOB-related genera (for example Sulfitobacter, Marinosulfuromonas, Rhodobacter, Thioclava) unique to ALWC-suspected layers suggested an important energetic contribution of steel and inorganic and organic sulfur intermediates in sustaining a biologically dominated sulfur cycle and a biocorrosion process. Overall, the identification of specific members from Oscillatoriales known to participate in sulfur cycling and the identification of specific iron-oxidizers, suggested their cooperative role in sustaining a long-term low water corrosion process on harbour structures associated with past events of ALWC.

329B Finding microbial species: taxonomic unit discovery using SSU ribosomal RNA sequences at a global scale João Frederico Matias Rodrigues*, Christian von Mering University of Zürich, Switzerland

Culture-independent methods used in the study of microbial organisms have shown that microbial diversity is far greater than what was believed so far. With these methods many novel microbial species have been found. It has also been shown that many microbes thrive best in specialized communities of organisms.

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A fundamental problem of culture-independent methods, with strong implications for many other studies, is that no clear way of defining taxonomic units exists. Here we discuss the technical and fundamental limitations of current methods of defining OTUs and explore alternatives.

The best chance of finding meaningful OTUs will undoubtedly come from the analysis of the globally available evidence for each group of individual organisms. As a first step, we have therefore developed a pipeline that searches, filters and aligns all SSU ribosomal RNA sequences from public repositories such as Genbank, Trace, and SRA. More than ten million bacterial 16S rRNA sequences were extracted from these databases. Around one million long sequences (>900bp), and more than nine million shorter length sequences.

This extremely large and ever-increasing number of sequences poses a big computational challenge since the more exact clustering methods require a large amounts of computer memory and processing time. Because no solutions existed to compute OTUs with this number of sequences in practical times, we have developed and implemented several clustering methods as parallel distributed programs which scale up well to cope with the current and future number of sequences.

Conventional approaches to OTU definition use hierarchical clustering algorithms with fixed sequence identity cutoffs between 97% and 99% for the clustering. The need for an arbitrary identity threshold is not an optimal approach as the threshold for species level taxonomic definitions can vary between lineages.

Finally, we evaluated the types of artifacts caused by the conventional clustering approaches by generating sets of SSU sequences produced by simulated evolution. Preliminary results show that current clustering approaches are prone to underestimation or overestimation of the number of taxonomic units depending on the cluster distance definition used. Work is currently in progress to develop new clustering methods that are less prone to these biases.

330B Microbial community structure based on 16S rRNA and pmoA genes in the dissolved methane treatment reactors receiving UASB effluents Norihisa Matsuura*1, Masashi Hatamoto2, Takashi Yamaguchi2, Akiyoshi Ohashi1 1Hiroshima University, Japan, 2Nagaoka University of Technology, Japan

Methane is a greenhouse gas with a 25-fold greater effect on global warming than carbon dioxide, thus reduction of methane emission is significantly important. Methanotrophs are unique aerobic bacteria utilizing methane as their sole carbon and energy source. Bioreactors with methanotrophs have been developed to reduce methane emissions from landfills, manure storage facilities and anaerobic wastewater treatment processes. As one of the bioreactors, closed down-flow hanging sponge (DHS) reactor is proposed. We had operated a system consisting of two sequenced DHS reactors for one year in a sewage treatment plant. In the first reactor (for dissolved methane recovery under the high methane and low oxygen conditions), some dissolved methane of UASB effluents was recovered as useful gas comprising more than 30% methane concentrations by physical gasification based on the gas-liquid equilibrium. In the second reactor (for residual dissolved methane oxidation), about 30% of unrecovered dissolved methane was biologically oxidized under aerobic condition. The dissolved methane of the UASB reactor effluent was almost completely removed in the system. In this study, biomass samples taken from the each reactor in the summer and winter were investigated to analyze microbial community structures based on the bacterial 16S rRNA gene with primer pairs of EUB338f/1492r and the particulate methane monooxygenase (pmoA) gene targeting methanotrophs with primer pairs of A189f/mb661r. We also employed terminal restriction fragment length polymorphism (T-RFLP) based on pmoA gene to monitor changes in methanotrophic population. As a result of clone analysis based on 16S rRNA gene, the microorganisms mainly belonged to Proteobacteria, Bacteroidetes, Acidobacteria and Firmicutes phyla. Methanotrophs were detected in the second dissolved methane oxidation reactor and accounted for 4.3% of total clones in the summer and 18.1% in the winter. The methanotrophic clones were closely related to Methylomicrobium species in the summer and Methylocaldum, Methylosoma, Methylomonas, Methylocystis in the winter. To reveal the detail methanotrophic communities, we constructed pmoA gene clone libraries. Although both type I and type II methanotrophs were detected, and type I methanotrophs were dominant. A T-RFLP profile along the height of the two reactors revealed that type I methanotrophs increased in both reactors in the summer. On the other hand, the percentages of type II methanotrophs increased a little

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in the winter. The results suggest that temperature is important factor to change methanotrophic communities, compared to methane and oxygen concentrations.

331B Bacterial community diversity increases with soil organic matter decomposition: insights from a dual-stable isotope probing method Rebecca Mau*1, Cindy Liu1, Michael Dwan1, Egbert Schwartz1, Paul Dijkstra1, Lance Price2, Bruce Hungate1 1Northern Arizona University, USA, 2Translational Genomics Research Institute, USA

Ecological theory, including the roles of biological diversity in modulating ecosystem stability, functioning, and responses to disturbance, has been primarily developed at the macroscopic scale, but its application to the micro scale has been difficult to gauge. With the development of new molecular methods, such as pyrosequencing and stable isotope probing, study of microbial community dynamics and related functional consequences in soils is becoming possible. We investigated the structure of microbial communities associated with altered carbon cycling in response to fresh substrate amendments, focusing on a phenomenon known as priming. We hypothesized that the priming phenomenon elicits responses from two distinct groups of microorganisms, mediating so-called positive and negative priming, in which soils receiving glucose substrate amendments degrade more or less soil organic matter respectively than a non-amended control soil. In this study, we implemented a novel dual-stable isotope probing technique in which 13C-labeled glucose and 18O-labeled water were used in order to separate organisms into those growing on glucose (labeled with 13C and 18O), those growing, but not utilizing the added glucose (labeled with 18O only), and those not growing (unlabeled). Fragments of 16S rRNA genes in the labeled DNA were then pyrosequenced to investigate the bacterial community structure associated with changes in soil organic matter decomposition. After the initial glucose pulse, respired soil organic carbon significantly decreased relative to a non-amended control (negative priming). After seven weekly pulses of glucose though, decomposition of soil organic matter exceeded rates in the non-amended control (positive priming). The bacterial community composition of both the glucose-assimilating and non-glucose-assimilating groups associated with positive and negative priming was very different. A bacterial community of low diversity dominated by the genus Arthrobacter was associated with negative priming, while a highly diverse and more even community was associated with positive priming. One possible explanation for the negative priming and low diversity community is that bacteria that degrade native soil organic matter were poor competitors with bacteria growing on glucose initially. However, over time the competitive advantage of the initial responders decreased, allowing for a more genetically and metabolically diverse community better capable of soil organic matter degradation to develop. A more diverse bacterial community has a higher probability of containing a greater array of enzymes needed for soil organic matter degradation. Our work contributes to a better understanding of the connections between belowground community dynamics and ecological function as well as microbial community succession.

332B A novel fine-scale approach for sampling deep-sea Fe oxide-dominated hydrothermal vent microbial mats at Loihi Seamount Sean McAllister*1, Nam Siu2, Richard Davis3, Bradley Tebo3, Craig Moyer2 1University of Delaware, USA, 2Western Washington University, USA, 3Oregon Health & Science University, USA

The scale at which sampling can be carried out in the deep ocean has largely been determined by the limits of robotic instrument manipulation at depth. Bulk sampling, via push cores, suction, or scoop samplers, collects mat material from heterogeneous microbial communities living in environments variable not only laterally with respect to an active vent (direct versus diffuse vent flow), but also with respect to depth across the steep redox gradient separating reduced hydrothermal fluid from oxygenated seawater. While initially unavoidable, these kinds of sampling strategies can only go so far in describing the intricate microbial ecology interactions occurring at hydrothermal vents. For this reason, a syringe sampler was developed for this study to sample targeted observable mat morphologies of surface mat material at smaller spatial scales.

Twenty-five samples were collected from active, Fe oxide-dominated mats from several vent sites at Loihi Seamount, Hawaii, all approximately the same temperature. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of these samples showed three distinct clusters correlating with the

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mat morphology observed prior to collection. Quantitative-PCR (qPCR) primers were designed to detect the relative abundance of the marine iron-oxidizing Zetaproteobacteria, which ranged from 0.9% – 81.8% of the total bacterial community. Five samples were selected for clone library (CL) analysis to capture the breadth of the bacterial community diversity. (I) Cluster one yellow and orange curd samples were dominated by Zetaproteobacteria (qPCR ave. 56.3%; CL 97.9%) belonging to recently described Zetaproteobacteria operational taxonomic units (ZOTU) commonly found at Loihi Seamount—ZOTUs 1 and 2. (II) Cluster two yellow veiled mat samples were dominated by Chromatiales (Gammaproteobacteria; ave. 43.4%) and Zetaproteobacteria (qPCR ave. 20.5%; CL ave. 29.9%) from ZOTU 2. (III) Cluster three samples were all taken from streamers at or near vent sources. Two clone libraries were constructed from highly dissimilar communities, both dominated by sulfur cycling bacteria. One was dominated by Desulfurivibrio spp. (Deltaproteobacteria; 35.6%), Nitrospira (10.2%), sulfur-oxidizing Epsilonproteobacteria (10.2%), and Zetaproteobacteria (qPCR 16.5%; CL 9.2%) from ZOTU 13. The other was dominated by Thiomicrospira spp. (27.3%), other unclassified Gammaproteobacteria, and Zetaproteobacteria (qPCR 9.6%; CL 20.3%) from ZOTUs 1 and 6. The heterogeneous nature of hydrothermal vent environments is highlighted when considering that members from all three of these highly diverse clusters, including samples representative of the breadth of diversity found in cluster three, could be found within 30 cm of each other at a single vent. Clearly, bulk sampling, though useful as a first step, will not allow microbial ecologists to tease apart community interactions nor the complexities of metabolic function at deep-sea hydrothermal vents. Further efforts should be made to develop and improve technologies that will allow sampling at smaller spatial scales.

333B Deep sequencing of evolving populations in bacterial biofilms Kerensa McElroy*, Jerry Woo, Marwan Majzoub, Janice Hui, Staffan Kjelleberg, Scott Rice, Suhelen Egan, Torsten Thomas University of New South Wales, Australia

Bacterial communities growing as biofilms are subject to a distinct lifecyle, featuring initial surface attachment, microcolony formation and dispersal of cells. Bacterial biofilms are sometimes characterised by high levels of heritable phenotypic variants, presumably resulting from genetic diversification during the biofilm lifecyle. As biofilms are a favoured lifestyle of many environmental and pathogenic bacteria, identifying the evolutionary processes responsible for this diversification has important implications, both for our understanding of ecological processes, such as niche adaptation, and to clinically relevant questions, such as the evolution of antibiotic resistance.

Here, we use longitudinal genome-wide deep sequencing to reveal the underlying genetic structure of bacterial populations growing as biofilms, for the model organisms Phaeobacter gallaeciensis 2.10 (an abundant marine bacterium) and Pseudomonas aeruginosa 18A (a clinical Cystic Fibrosis isolate). Biofilms were grown under defined laboratory conditions known to generate reproducible phenotypic diversification. Samples from different stages of biofilm development were then sequenced to very high coverage (>800x). By accounting for sequencing errors using a matched-sample approach, variants with population frequencies as low as 0.5% could be accurately identified.

For P. gallaeciensis 2.10, four variants were identified (three nucleotide substitutions and one 1bp deletion), with population frequencies ranging from 0.5% to 20.0%, corresponding to the frequencies of morphological phenotypes. Notably, one variant present at 19.7% affected a transcriptional regulator (PgaR) that responds to the quorum-sensing molecule N-acyl homoserine lactone. PgaR was recently shown to control the production of the antibiotic tropodithietic acid, which is thought to give P. gallaeciensis 2.10 a competitive advantage against other bacteria when colonizing surfaces. The presence of PgaR mutants within the biofilm population therefore suggests that TDA production may have an associated cost, and that some cells ‘cheat’ by not manufacturing this possibly expensive secondary metabolite.

In the P. aeruginosa 18A biofilm, eight genetic variants were identified, with frequencies varying from 2.9% to 96.9%. Variants were either single nucleotide substitutions or small deletions. Affected genes included an alginate biosynthesis transcriptional activator, a chemotaxis response regulator, a rod-shape determining protein, a two-component system response regulator, an ABC transporter, and phosphomannomutase. The latter five genes were also affected by the seven variants observed in an independent replicate of this experiment, with three mutations being identical between the two

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replicates. The reproducibility of these results suggests that genetic variation arising during biofilm growth does not result from genetic drift, but rather from selection acting on specific genes.

In general, the extent and nature of genetic variation was comparable for biofilms of both model organisms, being driven by selection for a small number of non-synonymous variants within key genes involved in biofilm- and competition-related pathways. Our results also demonstrate that genome-wide deep sequencing can rapidly, accurately and comprehensively describe genetic variation within evolving populations.

334B Analysis of the bacterial diversity in the fecal material of the endangered Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis Richard McLaughlin*1, Q. Zhao2, M. Chen2, J. Zheng2, D. Wang2 1Gateway Technical College, United States, 2Institute of Hydrobiology, Chinese Academy of Sciences, China

The aim of this study was to determine the bacteria present in the fecal material of the endangered Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis. Fecal samples were collected from 12 Yangtze finless porpoises living in the wild at Poyang Lake, located in Jiangxi Province, China. To determine the bacterial diversity, a 16S rRNA gene clone library using the bacterial PCR primers fD1 and rP2, was prepared. A total of 138 near-full-length sequences were analyzed and 39 operational taxonomic units (OTUs) were identified. Sequences showing ≥97% similarity were grouped together as an OTU. Six different phyla were identified in which 38 OTUs were classified. Most of the OTUs contained sequences belonged to the phylum Firmicutes (51.3%), followed by Tenericutes (17.9%), Proteobacteria (15.4%), Actinobacteria (7.7%), Deinococcus-Thermus (2.6%) and Cyanobacteria (2.6%). A phylum could not be assigned for one clone within one OTU (2.6%). It appears that the Yangtze finless porpoise has a more diverse range of bacteria compared to other aquatic mammals, such as seals.

335B The Microbial Database for Danish Wastewater Treatment Plants Artur Mielczarek*, Poul Larsen, Jeppe Lund Nielsen, Per Halkjær Nielsen Aalborg University, Denmark

Activated sludge treatment plants are the most used wastewater treatment systems worldwide for biological nutrient removal (N and P) and P-recovery. In order to ensure optimal operation, process optimization and trouble-shooting, it is crucial to understand the structure and function of the microbial communities and the factors controlling their composition and activity. At Aalborg University we have set up the project "Microbial Database for Danish Wastewater Treatment Plants" that regularly collect data from more than 30 full-scale biological nutrient removal treatment plants from all around Denmark. The aim is to learn more about the microbial communities, the factors that control these and better ways to obtain a stable and sustainable operation of the full-scale wastewater treatment plants.

Activated sludge samples were collected four times a year for seven treatment plants (January, May, August, November) and for the remaining plants twice a year (January, August). A full process description of the key treatment plant process parameters was collected and compared to a visual light microscopical characterization and a quantitative analysis by fluorescence in situ hybridization using more than 35 specific oligonucleotide probes. Relations between the plants and their operation was then analysed by a range of statistical methods i.e. MANOVA, PCA and cluster analysis.

The microbial populations in Danish full-scale activated sludge wastewater treatment plants with nutrient removal revealed the presence of a so-called "Danish standard activated sludge" for this type of plants. The key functional groups have been identified and they constituted up to 80% of total biomass. It was possible to identify a group of core microorganisms that is common for all investigated Danish plants. Their seasonal population variations were analysed and it was demonstrated that they were relatively stable during the time of investigation and similar between the plants. In spite of the population's similarity, it has been proven by statistical measures that the presence of two functional groups, namely Polyphosphate Accumulating Organisms/Glycogen Accumulating Organisms (PAO/GAO) and filamentous bacteria, in each treatment plant had its own unique microbial fingerprint.

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The Microbial Database has proven to be a powerful tool for exploration of the complex activated sludge ecosystems. It has provided a great insight in the typical population structure in a range of different nutrient removal plants and shown relative stable communities with minor seasonal and yearly variations.

336B Molecular diversity of the giant symbiotic bacterium Epulopiscium fishelsoni in fish from the Red Sea Sou Miyake*, Uli Stingl KAUST, Saudi Arabia

Epulopiscium fishelsoni ('Epulos') are unusual intestinal symbionts that inhabit the intestinal tracts of certain surgeonfish species. Originally described as Protists (Fishelsen et al., 1985) and discovered in fish guts from the Gulf of Aqaba, Red Sea, these Gram-positive Clostridia-related Bacteria can reach sizes well beyond the commonly observed prokaryotic size classes (in excess of 600 µm). Epulos are not only unusual in size, but also in having a complex lifecycle that includes the release of living daughter cells from a mother cell, extreme polyploidy (>100,000 genome copies), and possessing an unusual distribution of packed DNA at the inside of the cytoplasmic membrane. Several morphotypes (A - J) of giant bacteria resembling Epulos have been identified based on light microscopy analyses, but phylogenetic information on the diversity of the Epulo community within a single gut, as well as of communities of different fish species is scarce. Previous 16S rRNA study revealed 7 different genetic clustering (<97% sequences identity) within the polyphyletic Epulo group (termed A1, A2, B, C1, C2, J1 and J2 respectively to correspond with the described morphotypes), but the study was limited to three species of Surgeonfish (Acanthurus nigrofuscus, Naso lituratus and Naso tonganus) from Hawaii, the Great Barrier Reef, and the Red Sea (Angert et al., 1993; Flint et al., 2005).

This study aims to reveal the co-evolutionary relationships between Epulos and host Surgeonfish species from the Red Sea. In this study, we investigated the Epulo diversity from the majority of surgeonfish found in the Red Sea, including Acanthurus sohal, A. gahhm, A. nigrofuscus, Ctenochaetus striatus, Naso elegans, N. hexacanthus, N. brevirostris, N. unicornis, Zebrasoma desjardinii and Z. xanthurum. Species-specific 16S rRNA genetic analysis of Epulos from intestinal tracts of the different fish were compared with mitochondrial cytochrome B sequences from the host to address potential co-evolutionary relationships between the host and the symbiont. Fluorescent in situ hybridization (FISH) and light microscopy was used to further visualize giant Epulo-like bacteria.

The study revealed that the Epulo diversity is much higher than anticipated, both within a single fish gut and in different fish species. We postulate that these novel clades also correspond to novel morphotypes. Additionally, the presence or absence of Epulos in surgeonfish seems to be related to feeding habits rather than the phylogeny of the host. By comparing the phylogenies of host and symbiont, we did not find clear patterns of co-evolution, and thus hypothesize that the symbionts are transmitted horizontally rather than vertically.

337B Microplanktonic community structure in a coastal system relative to a Phaeocystis bloom inferred from morphological and Tag pyrosequencing methods Sébastien Monchy1, Jean-David Grattepanche1, Elsa Breton1, Dionigia Meloni2, Giovanna Sanciu2, Magali Chabé2, Laurence Delhaes2, Eric Viscogliosi*2, Télesphore Sime-Ngando3, Urania Christaki1 1LOG, Université du Littoral Côte d'Opale, France, 2CIIL, Institut Pasteur de Lille, France, 3LMGE, Université Blaise Pascal, France

Massive phytoplankton blooms, like the recurrent Phaeocystis proliferation observed every year in the Eastern English Channel (EEC), have a significant influence on the overall planktonic community structure and their food web dynamics. As well as being an important area for local fisheries, the EEC is an ideal ecosystem for work on microbial diversity. This is because, although its environmental context is relatively complex, it is reasonably well understood due to several years of monitoring and morphological observation of its planktonic organisms. The objective of our study was to better understand the under-explored microbial eukaryotic diversity relative to the Phaeocystis bloom.

The community structure of microplankton (diatoms, haptophytes, ciliates and dinoflagellates) was studied through morphological observations and tag pyrosequencing.

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During the annual Phaeocystis spring bloom, the phytoplankton biomass increased by 34 fold, while the microzooplankton biomass showed a 4 fold increase, representing on average about 4.6% of the biomass of their phytoplankton prey. Tag pyrosequencing unveiled an extensive diversity of Gymnodiniaceae, with G.spirale and G.fusiformis representing the most abundant reads. An extended diversity of Phaeocystales, with partial 18S rDNA genes sequence identity as low as 85% was found, with taxa corresponding to P.globosa, but also to unknown Phaeocystaceae.

Morphological analyses and pyrosequencing were generally in accordance with capturing frequency shifts of abundant taxa. Tag pyrosequencing allowed highlighting the maintenance of microplankton diversity during the Phaeocystis bloom and the increase of the taxa presenting low number of reads (minor taxa) along with the dominant ones in response to biotic and/or abiotic changing conditions. Although molecular approaches have enhanced our perception on diversity, it has come to light that the challenge of modelling and predicting ecological change requires the use of different complementary approaches, to link taxonomic data with the functional roles of microbes in biogeochemical cycles.

338B Dynamics changes in soil microbial community inoculated with phosphate-solubilizing bacteria (PSB) for enhancing Cd removal efficiency in phytoremediation Hee Sun Moon*, Seulki Jeong, Kyoungphile Nam Seoul National University, South Korea

Phytoextraction has demonstrated its beneficial use in the removal of heavy metals from polluted soil. Many researchers have focused on plant growth promoting rhizobacteria (PGPR) for enhancing biomass production as well as tolerance of the plants to heavy metals during phytoremediation. In particular, phosphate-solubilizing bacteria (PSB), one type of the PGPR, are effective in releasing phosphorous for plant growth through solubilization and these bacterial activities can affect phytoavailability in soils. This study was conducted to investigate the activity of inoculated PSB in soil for enhancing phytoextraction of Cd, and the microbial community dynamic during phytoextraction. Phytoextraction was carried out using Brassica juncea in artificially contaminated soil with Cd (38.8±1.45 mg/kg), and Bacillus megaterium(DSM No. 3228) as PSB were used. Each pot was inoculated with B. megaterium after transplanting and re-inoculated on the 1st, 2nd, 4th and 6th week to maintain a sufficient bacterial population. Uninoculated pot was used as control. The plants were harvested on the 1st, 2nd, 4th, 6th and 8th week to measure Cd accumulation in plants. For analysis of soil microbial community, DNA was extracted from soil, and used for PCR. Pyrosequencing was performed on a 454 Life Sciences Sequencer FLX machine (Roche, Broma, Sweden). With B. megaterium, the total dry weight of plants significantly increased from 10.3 mg to 101.0 mg up to the 6th weeks. The total plant dry weight of 295.6 mg was observed on the 8th week while it was 65.8 mg in the uninoculatd soil. Also, the Cd accumulation in plants with B. megaterium inoculation was greater by more than two times after 6 weeks when compared with the uninoculated control. These indicated that B. megaterium inoculation enhanced the plant growth and increased the bioavailabilty of Cd as well. During the phytoextraction, the soil pH with B. megaterium was slightly decreased from 5.78 to 4.91, and this may result from the secretion of organic acid such as IAA (Indole-3-acetic acid) by B. megaterium. Pyrosequencing analysis of soil was conducted for comparison of microbial community structure in soil with or without bacterial inoculation. Mesorhizobium sp. and Pseudonocardia sp. including Bacillus sp. were observed throughout the growing period (8 weeks) in soil. Prosthecomicrobium sp, Gallionella sp., and Filomicrobium sp., appeared in soil without inoculation while some bacteria such as Methylobacterium sp. and Micromonosporaceae sp. disappeared. Also, Nocardioidaceae sp., and Altererythrobacter sp. disappeared in soil with inoculation. Interestingly, B. megaterium (92% similarity) was detected only after 8 week in the inoculated soil, and Bacillus sp. accounted for more than half of the total bacteria community in soil, which resulted in the increase in the dominance (Simpson index: 0.148) of the community and the decrease in the richness and diversity (Shannon index: 3.835) compared to the other soils. The findings will enhance the efficiency of phytoremediation using the PSB application to heavy metal-contaminated soil. Furthermore, these results may provide the information on microbial processes and their activity in soil during phytoremediation.

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339B Microbial diversity in the frass of an endemic New Zealand wood boring beetle larvae, the Huhu grub (Prionoplus reticularis, Coleoptera; Cerambycidae) Hugh Morgan*, Ian McDonald, Jay Viswam University of Waikato, New Zealand

Efficient lignocellulose conversion to fermentable sugars presents the major challenge to the utilisation of these substrates for biofuels, but removing the lignin "glue" is thought to be the rate limiting step in this process. Many insects degrade woody substrates as their main dietary substrate with a consequent de-lignification during digestion.The grub of the indigenous Huhu beetle preferentially matures on decaying softwood timber accumulating a partially de-lignified frass in the tunnels it bores.

We have cultured grubs captured from the wild on either softwood or cellulose diets for periods of over 6 months to allow the gut microflora to adapt to these substrates. We have then cultured organisms and extracted DNA from the frass which we collect from these animals. We hypothesise that the gut microbiome of grubs on the softwood (lignocellulose) diet will be enriched for lignin metabolising microbes compared to grubs raised on a cellulose diet, and that these differences will be manifest in the frass flora. Fungal diversity in DNA extracted from the frass has been characterised by clone library analysis of the amplified ITS region, while bacterial diversity has been accessed by 16S sequence diversity. Trichoderma, Pichia, Candida and uncultured basidiomycete species dominate the softwood derived frass. Candida shehatae is a prevalent member of the community, and also occurs in cellulose derived frass. Type cultures of this species are recorded as degrading xylan but not cellulose, so the prevalence in the frass was surprising.We have cultured C. shehatae from frass and will report on its ability to utilise cellulose and lignocellulose substrates.

NMR analysis of softwood frass indicates greater change in carbohydrate rather than lignin compositon. This can be interpreted as indicating that either the gut microbiome is not utilising lignocellulose per se but merely scavenging polysaccharides either loosely attached or freed due to chewing, or that a brown rot Postia-type degradation is evident, where access to wood polysaccharides without major disruption of lignin can occur.

340B Community compositions in swine manure biogas plants Freya Mosbæk*, Piotr Starnawski, Jeppe Lund Nielsen Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, Denmark

The conversion of biomass into bioenergy has become an increasingly important alternative to fossil fuel, and several studies have been conducted to describe the diversity in the digesters. Most of these have been based on total extraction of DNA for downstream phylogenetic analysis without distinguishing the origin of the DNA. However, a large part of the extractable DNA derives from partial or completely lysed cells indigenous to the feeding material, which in most cases are of little interest for understanding the processes taking place in the digester. For this reason we used propidium monoazide treatment on samples from 9 different biogas plants prior to DNA extraction in order to avoid the impact of DNA from semi-digested and lysed cells as well as extracellular DNA. Propidium monoazide (PMA) will bind to free DNA upon a photo catalyzed reaction and the PMA-DNA complex will become inaccessible for amplification by PCR. Therefore only DNA from intact cells will be amplified.

The core microbial populations from the 9 biogas plants were identified in-depth using Illumina Hiseq 2000 amplicon sequencing. DNA extracted from samples, using different extraction protocols were treated with PMA and compared with the results of non-treated samples.

Using Qiime and MEGAN for data analysis, a significant different and less diverse microbial consortium was found in the two treatments. We observed a few reduced phyla in the PMA treated samples compared to the non-treated samples. In the non-treated samples a large amount of Proteobacteria especially Pseudomonadales was detected, which was not observed in the PMA treated samples. This could indicate that the PMA treatment is a resourceful means to remove extracellular DNA and DNA from lysed cells.

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A large part of the intact cells in the systems were identified as Firmicutes and Actinobacteria. Bacteria from these two phyla are known to be part of the biological processes occurring in biogas plants. We observed a huge group of unclassified sequences during the Qiime analysis. These sequences were afterward identified using RDP classifier and MEGAN and shown to predominantly belong to Bacteroidetes. The in-depth analyzed composition of bacterial phyla is generally in accordance with previous studies, although in different proportions and with reduced diversity.

341B Microbial diversity pattern during the cheese ripening: study on traditional Croatian cheeses Mirna Mrkonjic Fuka*1, Marion Engel2, Stefanie Wallisch2, Andrea Skelin1, Sulejman Redžepović1, Michael Schloter2 1University of Zagreb Faculty of Agriculture, Croatia, 2Helmholtz Zentrum München, Germany

Cheese-ripening is a time consuming process which involves series of biochemical reactions mediated by microorganisms. The diversity and interactions of the cheese microbiota contributes greatly to the complexity of the cheese-ripening process, and it is crucially important for developing the unique sensory characteristics of each traditional cheese variety. Therefore an in depth understanding on the microflora during cheese ripening is highly needed for understanding its role in cheese (eco) system and for the improvement (when needed) of organoleptic properties of cheeses.The aim of this study was to investigate the dynamic of bacterial communities of three traditional Croatian raw milk cheeses and to identify the dominant species based 16S rRNA gene barcoding 454 pyrosequencing and DGGE during cheese ripening. To achieve this aim milk and cheese samples from three different types of raw ewe’s milk cheeses produced in Mediterranean area of Croatia were collected. The raw milk, raw cheese and cheese samples during the ripening were sampled. We hypothesize that: (1) the dominant population will be specific for each cheese type, (2) the most of the variability in the community structure will be present during the initial stages of ripening and (3) the community will be stabilized within the first 30 days of ripening. Surprisingly, differences in bacterial diversity pattern at the same ripening stage between different farms of the same cheese type investigated were comparable low. Although considerable differences were noticed among different farms or ripening stages similar patterns and recurrent sequences were also observed. During initial stages of ripening the strong presence of sequences related to Enterobacteriacae were noticed. During the ripening the number of sequences related to lactic acid bacteria (LAB) increases and the abundance of sequences related to gram negative food spoilage or potentially pathogenic species rapidly decreases. Whereas fingerprinting patterns were highly dynamic during the initial ripening phases, after 60 days of ripening overall bacterial community composition was very stable. Sequence analysis revealed dominance of Lactococcus lactis spp. in most samples and strong presence of Enterococcus spp. or Streptococcus spp. depending on each cheese type.

342B Investigation of microbial diversity of aerosols at a saline playa in Victoria, Australia using culturing and 454 sequencing Chris Munday*1, Patrick De Deckker1, Nigel Tapper2, Gwen Allison1 1Australian National University, Australia, 2Monash University, Australia

Global dust emissions have increased over recent decades, and represent a mechanism for the transport of various factors including microorganisms. Major dust storms have occurred in Australia over the past ten years affecting large parts of the continent including the heavily populated east coast. The drought has also had extensive regional affects. Lake Gnarpurt is a saline lake located in volcanic plains 130km West of Melbourne. Lake Gnarpurt has dried out in the past decade due to prolonged drought and is a significant source of airborne dust in the local area. The dust has adversely affected farmland, and has the potential to affect nearby population centres with anecdotal reports of illness to residents.

The aim of this study was to utilize high throughput 16S rRNA sequencing (HTS) and traditional culture based analysis to characterize the microflora of aerosols, water and sediment collected from Lake Gnarpurt during the summers of 2008, 2009 and 2010, and the winter of 2008.

Aerosol samples were collected on cellulose nitrate filters attached to a portable vacuum pump fixed at various heights to a tethered balloon. Sediment, and when applicable, water samples were aseptically collected from the lake surface. Total DNA was extracted using MoBio PowerWater (water and filter

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samples) or UltraClean (sediment) samples and the V1-V3 region of the 16S rRNA was amplified and sequenced using HTS 454 titanium sequencing. Sequences were analyzed using the Mothur pipeline. Bacteria were propagated using tryptic soy agar, colonies were purified, and identified using 16S rRNA sequencing.

An average of nearly 8,800 high quality sequences were obtained for each sample, with preliminary estimates of diversity showing that water samples were more diverse than sediments. Diversity indices varied for aerosols depending on meteorological conditions, however diversity generally decreased with increasing sampling height.

Collectively, representatives from at least 16 different phyla were identified. The most common phylum was Proteobacteria, representing approximately one third of all sequences. Multiple genera typical of human pathogens were present as a minor component of the microflora (<150 reads/sample). The microflora composition of the different samples was compared using distance matrices Jaccard and Yue & Clayton theta. Water samples collected during winter and multiple aerosol samples collected at 1m simultaneously formed separate clusters in both matrices. There was great variability in the microflora of aerosol samples collected at different heights and times.

A much simpler microflora was characterized using traditional culture methods. Cultured isolates (n=270) were identified and assigned to 7 different phyla and 15 different families. Firmicutes were the dominant phylum, with the Bacillaceae the most dominant family overall. The overall taxonomic diversity was similar between sediments and aerosols, however diversity decreased with increasing height for aerosol samples.

These results demonstrate the advantage of using HTS sequencing to characterize the microflora of environmental samples, particularly aerosols. Despite the proximity of the source sample to the collection sites, the data indicates that the microflora of aerosols above Lake Gnarpurt vary greatly over time and at different heights, perhaps suggesting a regional, rather than local influence.

tuesday 21 august ps14 – microbial community diversity ...llebbe/abstracts/isme 14 abstract...

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