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      Anaerobic Oxidation of Methane at a Marine Methane Seep in a Forearc Sediment Basin off Sumatra, Indian Ocean

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          Abstract

          A cold methane seep was discovered in a forearc sediment basin off the island Sumatra, exhibiting a methane-seep adapted microbial community. A defined seep center of activity, like in mud volcanoes, was not discovered. The seep area was rather characterized by a patchy distribution of active spots. The relevance of anaerobic oxidation of methane (AOM) was reflected by 13C-depleted isotopic signatures of dissolved inorganic carbon. The anaerobic conversion of methane to CO 2 was confirmed in a 13C-labeling experiment. Methane fueled a vital microbial community with cell numbers of up to 4 × 10 9 cells cm −3 sediment. The microbial community was analyzed by total cell counting, catalyzed reporter deposition–fluorescence in situ hybridization (CARD–FISH), quantitative real-time PCR (qPCR), and denaturing gradient gel electrophoresis (DGGE). CARD–FISH cell counts and qPCR measurements showed the presence of Bacteria and Archaea, but only small numbers of Eukarya. The archaeal community comprised largely members of ANME-1 and ANME-2. Furthermore, members of the Crenarchaeota were frequently detected in the DGGE analysis. Three major bacterial phylogenetic groups (δ - Proteobacteria, candidate division OP9, and Anaerolineaceae) were abundant across the study area. Several of these sequences were closely related to the genus Desulfococcus of the family Desulfobacteraceae, which is in good agreement with previously described AOM sites. In conclusion, the majority of the microbial community at the seep consisted of AOM-related microorganisms, while the relevance of higher hydrocarbons as microbial substrates was negligible.

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          16S ribosomal DNA amplification for phylogenetic study.

          A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.
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            A microbial consortium couples anaerobic methane oxidation to denitrification.

            Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale. Freshwater sediments often receive increased downward fluxes of nitrate from agricultural runoff and upward fluxes of methane generated by anaerobic decomposition. In theory, prokaryotes should be capable of using nitrate to oxidize methane anaerobically, but such organisms have neither been observed in nature nor isolated in the laboratory. Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate. Here we show that the direct, anaerobic oxidation of methane coupled to denitrification of nitrate is possible. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitrification in the complete absence of oxygen. This consortium consisted of two microorganisms, a bacterium representing a phylum without any cultured species and an archaeon distantly related to marine methanotrophic Archaea. The detection of relatives of these prokaryotes in different freshwater ecosystems worldwide indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles.
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              Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set.

              The design and evaluation of a set of universal primers and probe for the amplification of 16S rDNA from the Domain Bacteria to estimate total bacterial load by real-time PCR is reported. Broad specificity of the universal detection system was confirmed by testing DNA isolated from 34 bacterial species encompassing most of the groups of bacteria outlined in Bergey's Manual of Determinative Bacteriology. However, the nature of the chromosomal DNA used as a standard was critical. A DNA standard representing those bacteria most likely to predominate in a given habitat was important for a more accurate determination of total bacterial load due to variations in 16S rDNA copy number and the effect of generation time of the bacteria on this number, since rapid growth could result in multiple replication forks and hence, in effect, more than one copy of portions of the chromosome. The validity of applying these caveats to estimating bacterial load was confirmed by enumerating the number of bacteria in an artificial sample mixed in vitro and in clinical carious dentine samples. Taking these parameters into account, the number of anaerobic bacteria estimated by the universal probe and primers set in carious dentine was 40-fold greater than the total bacterial load detected by culture methods, demonstrating the utility of real-time PCR in the analysis of this environment.
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                Author and article information

                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbio.
                Frontiers in Microbiology
                Frontiers Research Foundation
                1664-302X
                21 December 2011
                2011
                : 2
                : 249
                Affiliations
                [1] 1simpleFederal Institute for Geosciences and Natural Resources Hannover, Germany
                [2] 2simpleFaculty of Natural Sciences, Leibniz Universität Hannover Hannover, Germany
                Author notes

                Edited by: Andreas Teske, University of North Carolina at Chapel Hill, USA

                Reviewed by: Nils Risgaard-Petersen, Aarhus University, Denmark; Heide N. Schulz-Vogt, Max-Planck-Institute for Marine Microbiology, Germany

                *Correspondence: Martin Krüger, Federal Institute for Geosciences and Natural Resources, Stilleweg 2, 30655 Hannover, Germany. e-mail: martin.krueger@ 123456bgr.de

                Present address: Barbara Teichert, Institut für Geologie und Paläontologie, Universität Münster, 48149 Münster, Germany.

                This article was submitted to Frontiers in Extreme Microbiology, a specialty of Frontiers in Microbiology.

                Article
                10.3389/fmicb.2011.00249
                3245565
                22207865
                731cce83-a0e1-4655-9b56-00647fb22aed
                Copyright © 2011 Siegert, Krüger, Teichert, Wiedicke and Schippers.

                This is an open-access article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.

                History
                : 09 August 2011
                : 23 November 2011
                Page count
                Figures: 7, Tables: 4, Equations: 0, References: 96, Pages: 16, Words: 12356
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                card–fish,hydrocarbon-dependent methanogenesis,stable isotopes,methane seep,dgge,quantitative pcr,aom

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