VAAM-Jahrestagung 2012 18.–21. März in Tübingen

137OTV028Testing the limits of 454 pyrotag sequencing: reproducibilityand quantitative assessmentG. Pilloni, M. Granitsiotis, T. Lueders*Helmholtz Zentrum München, Institute of Groundwater Ecology,Neuherberg, GermanyCharacterization of microbial community structure via 16S rRNA geneprofiling has been greatly advanced in recent years by the application ofamplicon pyrosequencing. The possibility of barcode “tagging”sequencing templates gives the opportunity to massively screen multiplesamples from environmental or clinical sources for community details.However, an on-going debate questions the reproducibility and semiquantitativerigour of pyrotag sequencing, and, as in the early days ofgenetic community fingerprinting, pros and cons are continuouslyprovided.In this study we investigate the reproducibility of bacterial 454 pyrotagsequencing over biological and technical replicates of natural microbiota.Moreover, via quantitatively defined template spiking to the naturalcommunity, we explore the potential for recovering specific templateratios within complex microbial communities. For this reason, we pyrotagsequenced three biological replicates of three samples, each belongingfrom yearly sampling campaigns of sediment from a tar oil contaminatedaquifer in Düsseldorf, Germany. Furthermore, we subjected one DNAextract to replicate technical analyses as well as to increasing ratios (0, 0.2,2 and 20%) of 16S rRNA genes from a pure culture (Vibrio fisheri)originally not present in the sample.Unexpectedly, taxa abundances were highly reproducible in our hands,with max standard deviation of 4% abundance across biological and 2%for technical replicates. Furthermore, our workflow was also capable ofrecovering V. fisheri amendmend ratios in reliable amounts (0, 0.29, 3.9and 23.8%). These results highlight that pyrotag sequencing, if done andevaluated with due caution, has the potential to robustly recapture taxatemplate abundances within environmental microbial communities.OTV029Microbial Communities of Marine Methane Seeps: Sketchingthe Big PictureS.E. Ruff* 1 , J. Biddle 2 , A. Teske 3 , A. Ramette 1 , K. Knittel 4 , A. Boetius 11 Max Planck Institute for Marine Microbiology, HGF MPG Group forDeep Sea Ecology and Technology, Bremen, Germany2 University of Delaware, College of Earth, Ocean and the Environment,Lewes, DE, USA, United States3 University of North Carolina at Chapel Hill, Department of MarineSciences, Chapel Hill, NC, USA, United States4 Max Planck Institute for Marine Microbiology, Department of MolecularEcology, Bremen, GermanyGlobal ocean sampling efforts like the GOS expedition [1] and theInternational Census of Marine Microbes (ICoMM) [2] have revealeddistinct microbial communities in surface and deep waters, coastal andopen ocean ecosystems as well as in pelagic and benthic realms [3] . Thispresentation aims at sketching the big picture of archaeal and bacterialcommunities inhabiting cold seeps. We have analyzed 26 methane seepecosystems of different temperature regimes across all major oceans fromthe Arctic to Antarctica. To identify the full range of residentmethanotrophic key players as well as microbial taxa within the rarebiosphere, 454-pyrosequencing of the variable region V6 within the 16SrRNA gene was applied. In addition to a description of biogeography,community composition, ß-diversity and covariation of certain taxa,environmental data were included in order to explain some of the emergingpatterns. First results indicate that the seep communities are far morediverse and distinct than previously assumed. 80% of the archaeal OTUs(operational taxonomic units at a 97% nucleotide similarity cut-off)belonged to a variable community occurring at some seeps, but not atothers. Interestingly, this variable community included all anaerobicmethanotrophic (ANME) key players with the ANME-2a/2b clade beingmost widespread. Around 18% of archaeal OTUs were unique (occurringonly at one seep) and only 2% of archaeal OTUs were residents (occurringat all seeps). These residents were identified as organisms of theMiscellaneous Crenarchaeotal Group and Marine Benthic Group B. Thebacterial rare biosphere was even more prominent with 30% of all bacterialOTUs being unique and only about 1% of resident OTUs. The remaining69% of all OTUs, the bacterial variable community, were dominated byDelta- and Gammaproteobacteria, while most of the bacterial residents areyet unknown, since they could merely be classified to the order level andlacked cultivated representatives. This presentation will discuss ways todefine core microbial communities of marine methane seeps in distinctocean realms and main factors driving their diversity.1. Nealson, K.H. and J.C. Venter (2007) "Metagenomics and the global ocean survey: what's in itfor us, and why should we care?" ISME J. 1: p. 185-187.2. http://icomm.mbl.edu/3. Zinger, L. et al. (2011) "Global Patterns of Bacterial Beta-Diversity in Seafloor and SeawaterEcosystems." PLoS ONE. 6: p. e24570.OTV030Prokaryotic diversity in Pacific Ocean manganese nodulesM. Blöthe*, A. SchippersBundesanstalt für Geowissenschaften und Rohstoffe, Geomicrobiology,Hannover, GermanyDNA extraction from different parts (hydrogenetic, diagenetic, core, edge)of a Pacific Ocean manganese nodule and a manganese crust collectedduring the cruise SO205 in 2010 with the German research vessel Sonnewere analyzed with qPCR and via clone libraries for 16S rRNA genes ofArchaea and Bacteria. Results indicate highest cell numbers in thediagenetic and circular edge nodule parts with about 1.7×10 8 - 3×10 8cells/g and similar values (1×10 8 cells/g) were obtained for the top 2 cm ofthe nodule surrounding sediment. Cell numbers inside the nodule and inthe hydrogenetic part were lower by one order of magnitude (about 10 7cells/g). Bacterial cell numbers were always higher than numbers ofArchaea. The diversity (Yue & Clayton Q YC similarity coefficient) ofbacterial and archaeal communities associated with the nodules wasdifferent from the community diversity in the sediment and on themanganese crust. Bacterial species highly similar to Shewanella benthicawere found in all clone libraries from the nodule but not in the surroundingsediment or in the manganese crust. Nearly all obtained archaeal 16SrRNA gene sequences belonged to the Marine Group I Crenarchaeota.OTV031Inhibition of heterotrophic bacteria by solar radiation in a humiclakeS. Glaeser 1,2 , F. Leunert 3 , I. Salka 3 , H.-P. Grossart 3 , *J. Glaeser 11 Justus LiebigUniversität, Mikrobiologie und Molekularbiologie, Gießen, Germany2 Justus Liebig Universität, Institut für Angewandte Mikrobiologie, Gießen,Germany3 Institut für Gewässerökologie und Binnenfischerei, Limnologie GeschiteterSeen, Stechlin, GermanyLight excitation of colored dissolved organic matter (cDOM) lead tophotochemical reactions that produce low molecular weight (LMW)growth substrates that stimulate bacterial activity and inhibitory reactiveoxygen species (ROS). In order to investigate the impact of ROSgeneration on bacterial activity we monitored diurnal cycles of ROSformation and bacterial activity in the humic south-west basin of LakeGrosse Fuchskuhle. High solar radiation caused strong inhibition ofbacterial 14 C-leucine and 14 C-acetate uptake in surface waters andincreased the fraction of membrane-damaged cells assessed by life/deadstaining. The inhibition was paralleled by the formation of ROS, whichvery likely are the agents causing bacterial inhibition. In order to verify ourdata, cultures representing predominant bacterial phylotypes of the SWbasin were incubated in the surface water layer by using dialysis bags.Acetate and leucine uptake and the fraction of membrane-damaged cellswere monitored in those cultures. Novosphingobium acidiphilum(Alphaproteobacteria) represents a persistent species of the SW basin andwas not hampered in activity by solar radiation. In contrast, the activity ofPolynucleobacter necessarius a predominant Betaproteobacteriarepresentative was strongly inhibited by high solar radiation as indicatedby a low uptake of acetate and leucine compared to early morning samples.Cultures of both strains showed a very high fraction of life cells that didnot decrease during daytime hours. Hence, we conclude that N.acidiphilum and P. necessarius have efficient mechanisms to cope withinhibitory products of photochemical reactions with respect tomaintenance of cell integrity. Interestingly, solar radiation mediatedformation of inhibitory substances leads to very low activity of P.necessarius, but not of N. acidiphilum. Hence, photochemical reactionsthat generate inhibitory ROS affect predominant bacteria of a humic lakein a species-specific manner.OTV032Phylogenetic characterization and comparison of microbialcommunities in mesophilic and thermophilic anaerobic digestersX. Dong*, M. Engel, M. SchloterHelmholtz Zentrum München, Environmental Genomics, Neuherberg,GermanyMesophilic (30-40°C) and thermophilic (45-60°C) anaerobic digestion ofsubstrate are the two main processes for biogas production. Mesophilicdigestion is the most commonly used process with higher operatingrobustness, while thermophilic digestion provides higher biogasproduction with improved hygiene by reducing the pathogens. The largelyunknown compositions of microbial communities, especially thehydrolytic bacterial communities involved in these processes are the key tounderstand the complex process. Thus cost-effective process could bechosen under different circumstances.Lab scale mesophilic (~38.8°C) and thermophilic (~55°C) digestersoperated with energy plants with semi-continuous stirring and dailyfeeding were used in this study. Bar-coded amplicon pyrosequencing ofBIOspektrum | Tagungsband 2012