The Experts below are selected from a list of 10362 Experts worldwide ranked by ideXlab platform
Michael W Friedrich - One of the best experts on this subject based on the ideXlab platform.
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identification of novel perchloroethene respiring microorganisms in anoxic river sediment by rna based stable isotope Probing
Environmental Microbiology, 2007Co-Authors: Sandra Kittelmann, Michael W FriedrichAbstract:Summary The halogenated compound tetrachloroethene (perchloroethene, PCE) is a persistent contaminant of aquifers, soils and sediments. Although a number of microorganisms are known to reductively dechlorinate PCE by dehalorespiration, their diversity and community structure especially in pristine environments remain elusive. In this study, we report on the detection of a novel group of dehalorespiring bacteria that reductively dechlorinate PCE to cisdichloroethene by RNA-based stable isotope Probing. Pristine river sediment was incubated at 15°C with PCE at low aqueous concentration. Upon formation of dechlorination products, the microbial community was probed with 13 C-labelled acetate as electron donor and carbon source. Terminal restriction fragment length polymorphism (T-RFLP) analysis of density-separated 16S rRNA revealed a predominantly 13 C-labelled bacterial population only in the microcosm with PCE in high-density gradient fractions, whereas in the control without PCE Bacteriaspecific rRNA was restricted to light gradient fractions. By cloning and sequence analysis of 16S rRNA, the predominant population was identified as a novel group of bacteria within the phylum Chloroflexi. These microorganisms, designated Lahn Cluster (LC), were only distantly related to cultivated dehalorespiring Dehalococcoides spp. (92‐94% sequence identity). Minor clone groups detected 13 C-labelled and thus, potentially involved in PCE dehalorespiration, were related to b-proteobacterial Dechloromonas spp., and d-Proteobacteria (Geobacteraceae, Desulfobacteraceae, Desulfobulbaceae). In contrast, clones from an ethene-producing microcosm incubated at 20°C grouped with known Dehalococcoides spp. Our results show that stable isotope Probing allows targeting dehalorespiring bacteria as functional guild, and to identify novel PCE-respiring populations previously not recognized.
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identification of acetate assimilating microorganisms under methanogenic conditions in anoxic rice field soil by comparative stable isotope Probing of rna
Applied and Environmental Microbiology, 2007Co-Authors: Tomoyuki Hori, Michael W Friedrich, Matthias Noll, Yasuo Igarashi, Ralf ConradAbstract:Acetate is the most abundant intermediate of organic matter degradation in anoxic rice field soil and is converted to CH4 and/or CO2. Aceticlastic methanogens are the primary microorganisms dissimilating acetate in the absence of sulfate and reducible ferric iron. In contrast, very little is known about bacteria capable of assimilating acetate under methanogenic conditions. Here, we identified active acetate-assimilating microorganisms by using a combined approach of frequent label application at a low concentration and comparative RNA-stable isotope Probing with 13C-labeled and unlabeled acetate. Rice field soil was incubated anaerobically at 25°C for 12 days, during which 13C-labeled acetate was added at a concentration of 500 μM every 3 days. 13C-labeled CH4 and CO2 were produced from the beginning of the incubation and accounted for about 60% of the supplied acetate 13C. RNA was extracted from the cells in each sample taken and separated by isopycnic centrifugation according to molecular weight. Bacterial and archaeal populations in each density fraction were screened by reverse transcription-PCR-mediated terminal restriction fragment polymorphism analysis. No differences in the bacterial populations were observed throughout the density fractions of the unlabeled treatment. However, in the heavy fractions of the 13C treatment, terminal restriction fragments (T-RFs) of 161 bp and 129 bp in length predominated. These T-RFs were identified by cloning and sequencing of 16S rRNA as from a Geobacter sp. and an Anaeromyxobacter sp., respectively. Apparently these bacteria, which are known as dissimilatory iron reducers, were able to assimilate acetate under methanogenic conditions, i.e., when CO2 was the predominant electron acceptor. We hypothesize that ferric iron minerals with low bioavailability might have served as electron acceptors for Geobacter spp. and Anaeromyxobacter spp. under these conditions.
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stable isotope Probing of microorganisms thriving at thermodynamic limits syntrophic propionate oxidation in flooded soil
Applied and Environmental Microbiology, 2004Co-Authors: Tillmann Lueders, Bianca Pommerenke, Michael W FriedrichAbstract:soil by rRNA-based Stable-Isotope Probing (SIP). After 7 weeks of incubation with [ 13 C]propionate (<10 mM) and the oxidation of 30 mol of 13 C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were 13 C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” 13 C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of 13 C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [ 13 C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.
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stable isotope Probing of rrna and dna reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil
Environmental Microbiology, 2003Co-Authors: Tillmann Lueders, Bianca Wagner, Peter Claus, Michael W FriedrichAbstract:Stable isotope Probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (< 22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of 'heavy' bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.
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enhanced sensitivity of dna and rrna based stable isotope Probing by fractionation and quantitative analysis of isopycnic centrifugation gradients
Environmental Microbiology, 2003Co-Authors: Tillmann Lueders, Mike Manefield, Michael W FriedrichAbstract:Stable isotope Probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.
Tillmann Lueders - One of the best experts on this subject based on the ideXlab platform.
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enrichment and characterization of a sulfate reducing toluene degrading microbial consortium by combining in situ microcosms and stable isotope Probing techniques
FEMS Microbiology Ecology, 2010Co-Authors: Petra Bombach, Tillmann Lueders, Antonis Chatzinotas, Thomas R Neu, Matthias Kastner, Carsten VogtAbstract:A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [13C]-α-toluene or [13C7]-toluene as growth substrates. Cells incubated with [13C]-α-toluene or [13C7]-toluene incorporated 8–15 at.%13C and 51–57 at.%13C into total lipid fatty acids, respectively, indicating a lower specific incorporation of 13C from [13C7]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [13C]-α-toluene and [13C7]-toluene into rRNA was analyzed by stable isotope Probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.
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dna stable isotope Probing
Nature Protocols, 2007Co-Authors: Andrew S. Whiteley, Bruce C. Thomson, Tillmann Lueders, Mike ManefieldAbstract:Stable-Isotope Probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-Isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4-5 days, with much of the time being committed to the ultracentrifugation step.
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stable isotope Probing of microorganisms thriving at thermodynamic limits syntrophic propionate oxidation in flooded soil
Applied and Environmental Microbiology, 2004Co-Authors: Tillmann Lueders, Bianca Pommerenke, Michael W FriedrichAbstract:soil by rRNA-based Stable-Isotope Probing (SIP). After 7 weeks of incubation with [ 13 C]propionate (<10 mM) and the oxidation of 30 mol of 13 C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were 13 C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in “heavy” 13 C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured “rice cluster I” lineage had incorporated substantial amounts of 13 C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [ 13 C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.
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stable isotope Probing of rrna and dna reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil
Environmental Microbiology, 2003Co-Authors: Tillmann Lueders, Bianca Wagner, Peter Claus, Michael W FriedrichAbstract:Stable isotope Probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (< 22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of 'heavy' bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.
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enhanced sensitivity of dna and rrna based stable isotope Probing by fractionation and quantitative analysis of isopycnic centrifugation gradients
Environmental Microbiology, 2003Co-Authors: Tillmann Lueders, Mike Manefield, Michael W FriedrichAbstract:Stable isotope Probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.
Colin J Murrell - One of the best experts on this subject based on the ideXlab platform.
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revealing the uncultivated majority combining dna stable isotope Probing multiple displacement amplification and metagenomic analyses of uncultivated methylocystis in acidic peatlands
Environmental Microbiology, 2008Co-Authors: Yin Chen, Nancy Stralispavese, Mark G. Dumont, Josh D. Neufeld, Levente Bodrossy, Niall P. Mcnamara, Nick Ostle, Maria J. I. Briones, Colin J MurrellAbstract:Summary Peatlands represent an enormous carbon reservoir and have a potential impact on the global climate because of the active methanogenesis and methan- otrophy in these soils. Uncultivated methanotrophs from seven European peatlands were studied using a combination of molecular methods. Screening for methanotroph diversity using a particulate methane monooxygenase-based diagnostic gene array revealed that Methylocystis-related species were dominant in six of the seven peatlands studied. The abundance and methane oxidation activity of Methy- locystis spp. were further confirmed by DNA stable- isotope Probing analysis of a sample taken from the Moor House peatland (England). After ultracentrifuga- tion, 13 C-labelled DNA, containing genomic DNA of
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stable isotope Probing implicates methylophaga spp and novel gammaproteobacteria in marine methanol and methylamine metabolism
The ISME Journal, 2007Co-Authors: Josh D. Neufeld, Rich Boden, Hendrik Schafer, Michael J Cox, Ian R Mcdonald, Colin J MurrellAbstract:Stable-Isotope Probing implicates Methylophaga spp and novel Gammaproteobacteria in marine methanol and methylamine metabolism
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identification of a complete methane monooxygenase operon from soil by combining stable isotope Probing and metagenomic analysis
Environmental Microbiology, 2006Co-Authors: Marc G Dumont, Stefan Radajewski, Ian R Mcdonald, Carlos B Miguez, Colin J MurrellAbstract:Stable isotope Probing (SIP) allows the isolation of nucleic acids from targeted metabolically active organisms in environmental samples. In previous studies, DNA-SIP has been performed with the one-carbon growth substrates methane and methanol to study methylotrophic organisms. The methylotrophs that incorporated the labelled substrate were identified with polymerase chain reaction and sequencing of 16S rRNA and 'functional genes' for methanotrophs (mxaF, pmoA, mmoX). In this study, a SIP experiment was performed using a forest soil sample incubated with (13)CH(4), and the (13)C-DNA was purified and cloned into a bacterial artificial chromosome (BAC) plasmid. A library of 2300 clones was generated and most of the clones contained inserts between 10 and 30 kb. The library was probed for key methylotrophy genes and a 15.2 kb clone containing a pmoCAB operon, encoding particulate methane monooxygenase, was identified and sequenced. Analysis of the pmoA sequence suggested that the clone was most similar to that of a Methylocystis sp. previously detected in this forest soil. Twelve other open reading frames were identified on the clone, including the gene encoding beta-ribofuranosylaminobenzene 5'-phosphate synthase, which is involved in the biosynthesis of the 'archaeal' C(1)-carrier, tetrahydromethanopterin, which is also found in methylotrophs. This study demonstrates that relatively large DNA fragments from uncultivated organisms can be readily isolated using DNA-SIP, and cloned into a vector for metagenomic analysis.
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stable isotope Probing linking microbial identity to function
Nature Reviews Microbiology, 2005Co-Authors: Marc G Dumont, Colin J MurrellAbstract:Stable isotope Probing (SIP) is a technique that is used to identify the microorganisms in environmental samples that use a particular growth substrate. The method relies on the incorporation of a substrate that is highly enriched in a stable isotope, such as (13)C, and the identification of active microorganisms by the selective recovery and analysis of isotope-enriched cellular components. DNA and rRNA are the most informative taxonomic biomarkers and (13)C-labelled molecules can be purified from unlabelled nucleic acid by density-gradient centrifugation. The future holds great promise for SIP, particularly when combined with other emerging technologies such as microarrays and metagenomics.
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identification of active methylotroph populations in an acidic forest soil by stable isotope Probing
Microbiology, 2002Co-Authors: Stefan Radajewski, James Ivor Prosser, David B Nedwell, Samantha A Morris, Donald S. Reay, Gordon Webster, Philip Ineson, Colin J MurrellAbstract:Stable-Isotope Probing (SIP) is a culture-independent technique that enables the isolation of DNA from micro-organisms that are actively involved in a specific metabolic process. In this study, SIP was used to characterize the active methylotroph populations in forest soil (pH 3·5) microcosms that were exposed to 13CH3OH or 13CH4. Distinct 13C-labelled DNA (13C-DNA) fractions were resolved from total community DNA by CsCl density-gradient centrifugation. Analysis of 16S rDNA sequences amplified from the 13C-DNA revealed that bacteria related to the genera Methylocella, Methylocapsa, Methylocystis and Rhodoblastus had assimilated the 13C-labelled substrates, which suggested that moderately acidophilic methylotroph populations were active in the microcosms. Enrichments targeted towards the active proteobacterial CH3OH utilizers were successful, although none of these bacteria were isolated into pure culture. A parallel analysis of genes encoding the key enzymes methanol dehydrogenase and particulate methane monooxygenase reflected the 16S rDNA analysis, but unexpectedly revealed sequences related to the ammonia monooxygenase of ammonia-oxidizing bacteria (AOB) from the β-subclass of the Proteobacteria. Analysis of AOB-selective 16S rDNA amplification products identified Nitrosomonas and Nitrosospira sequences in the 13C-DNA fractions, suggesting certain AOB assimilated a significant proportion of 13CO2, possibly through a close physical and/or nutritional association with the active methylotrophs. Other sequences retrieved from the 13C-DNA were related to the 16S rDNA sequences of members of the Acidobacterium division, the β-Proteobacteria and the order Cytophagales, which implicated these bacteria in the assimilation of reduced one-carbon compounds or in the assimilation of the by-products of methylotrophic carbon metabolism. Results from the 13CH3OH and 13CH4 SIP experiments thus provide a rational basis for further investigations into the ecology of methylotroph populations in situ.
Yue-qin Tang - One of the best experts on this subject based on the ideXlab platform.
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using dna based stable isotope Probing to reveal novel propionate and acetate oxidizing bacteria in propionate fed mesophilic anaerobic chemostats
Scientific Reports, 2019Co-Authors: Hui-zhong Wang, Min Gou, Yong Nie, Dan Zheng, Masaru K Nobu, Takashi Narihiro, Yue-qin TangAbstract:Propionate is one of the most important intermediates of anaerobic fermentation. Its oxidation performed by syntrophic propionate-oxidizing bacteria coupled with hydrogenotrophic methanogens is considered to be a rate-limiting step for methane production. However, the current understanding of SPOB is limited due to the difficulty of pure culture isolation. In the present study, two anaerobic chemostats fed with propionate as the sole carbon source were operated at different dilution rates (0.05 d−1 and 0.15 d−1). The propionate- and acetate-oxidizing bacteria in the two methanogenic chemostats were investigated combining DNA-stable isotope Probing (DNA-SIP) and 16S rRNA gene high-throughput sequencing. The results of DNA-SIP with 13C-propionate/acetate suggested that, Smithella, Syntrophobacter, Cryptanaerobacter, and unclassified Rhodospirillaceae may be putative propionate-oxidizing bacteria; unclassified Spirochaetaceae, unclassified Synergistaceae, unclassified Elusimicrobia, Mesotoga, and Gracilibacter may contribute to acetate oxidation; unclassified Syntrophaceae and Syntrophomonas may be butyrate oxidizers. By DNA-SIP, unclassified OTUs with 16S rRNA gene abundance higher than 62% of total Bacteria in the PL chemostat and 38% in the PH chemostat were revealed to be related to the degradation of propionate. These results suggest that a variety of uncultured bacteria contribute to propionate degradation during anaerobic digestion. The functions and metabolic characteristics of these bacteria require further investigation.
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identification of novel potential acetate oxidizing bacteria in thermophilic methanogenic chemostats by dna stable isotope Probing
Applied Microbiology and Biotechnology, 2019Co-Authors: Dan Zheng, Hui-zhong Wang, Min Gou, Yong Nie, Masaru K Nobu, Takashi Narihiro, Yue-qin TangAbstract:Syntrophic oxidization of acetate and propionate are both critical steps of methanogenesis during thermophilic anaerobic digestion. However, knowledge on syntrophic acetate-oxidizing bacteria (SAOB) and syntrophic propionate-oxidizing bacteria (SPOB) is limited because of the difficulty in pure culture isolation due to symbiotic relationship. In this study, two thermophilic acetate-fed anaerobic chemostats, ATL (dilution rate of 0.025 day−1) and ATH (0.05 day−1) and one thermophilic propionate-fed anaerobic chemostat PTL (0.025 day−1) were constructed, AOB and POB in these chemostats were studied via microbial community analysis and DNA Stable-Isotope Probing (SIP). The results showed that, in addition to Tepidanaerobacter, a known SAOB, species of Thauera, Thermodesulfovibrio, Anaerobaculum, Ruminiclostridium, Comamonas, and uncultured bacteria belonging to Lentimicrobiaceae, o_MBA03, Thermoanaerobacteraceae, Anaerolineaceae, Clostridiales, and Ruminococcaceae were determined to be potential AOB in chemostats. Pelotomaculum was the key SPOB detected in the propionate-fed chemostat. Based on the intense fluorescence of coenzyme F420, majority of Methanosarcina cells in acetate-fed chemostats were involved in hydrogenotrophic methanogenesis, suggesting the existence of highly active SAOB among the detected AOB. In the propionate-fed chemostat, most of the species detected as AOB were similar to those detected in the acetate-fed chemostats, suggesting the contribution of the syntrophic acetate oxidization pathway for methane generation. These results revealed the existence of previously unknown AOB with high diversity in thermophilic chemostats and suggested that methanogenesis from acetate via the syntrophic oxidization pathway is relevant for thermophilic anaerobic digestion.
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Identification of Novel Butyrate- and Acetate-Oxidizing Bacteria in Butyrate-Fed Mesophilic Anaerobic Chemostats by DNA-Based Stable Isotope Probing.
Microbial Ecology, 2019Co-Authors: Hui-zhong Wang, Ya-ting Chen, Min Gou, Zi-yuan Xia, Yong Nie, Yue-qin TangAbstract:Butyrate is one of the most important intermediates during anaerobic digestion of protein wastewater, and its oxidization is considered as a rate-limiting step during methane production. However, information on syntrophic butyrate-oxidizing bacteria (SBOB) is limited due to the difficulty in isolation of pure cultures. In this study, two anaerobic chemostats fed with butyrate as the sole carbon source were operated at different dilution rates (0.01/day and 0.05/day). Butyrate- and acetate-oxidizing bacteria in both chemostats were investigated, combining DNA-Stable Isotope Probing (DNA-SIP) and 16S rRNA gene high-throughput sequencing. The results showed that, in addition to known SBOB, Syntrophomonas, other species of unclassified Syntrophomonadaceae were putative butyrate-oxidizing bacteria. Species of Mesotoga, Aminivibrio, Acetivibrio, Desulfovibrio, Petrimonas, Sedimentibacter, unclassified Anaerolineae, unclassified Synergistaceae, unclassified Spirochaetaceae, and unclassified bacteria may contribute to acetate oxidation from butyrate metabolism. Among them, the ability of butyrate oxidation was unclear for species of Sedimentibacter, unclassified Synergistaceae, unclassified Spirochaetaceae, and unclassified bacteria. These results suggested that more unknown species participated in the degradation of butyrate. However, the corresponding function and pathway for butyrate or acetate oxidization of these labeled species need to be further investigated.
Mike Manefield - One of the best experts on this subject based on the ideXlab platform.
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identification of ciliate grazers of autotrophic bacteria in ammonia oxidizing activated sludge by rna stable isotope Probing
Applied and Environmental Microbiology, 2010Co-Authors: Ana Maria Moreno, Carsten Matz, Staffan Kjelleberg, Mike ManefieldAbstract:It is well understood that protozoa play a major role in controlling bacterial biomass and regulating nutrient cycling in the environment. Little is known, however, about the movement of carbon from specific reduced substrates, through functional groups of bacteria, to particular clades of protozoa. In this study we first identified the active protozoan phylotypes present in activated sludge, via the construction of an rRNA-derived eukaryote clone library. Most of the sequences identified belonged to ciliates of the subclass Peritrichia and amoebae, confirming the dominance of surface-associated protozoa in the activated sludge environment. We then demonstrated that 13C-labeled protozoan RNA can be retrieved from activated sludge amended with 13C-labeled protozoa or 13C-labeled Escherichia coli cells by using an RNA stable isotope Probing (RNA-SIP) approach. Finally, we used RNA-SIP to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions, respectively. RNA-SIP analysis revealed that the peritrich ciliate Epistylis galea dominated the acquisition of carbon from bacteria with access to CO2 under ammonia-oxidizing conditions, while there was no evidence of specific grazing on acetate consumers under denitrifying conditions.
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dna stable isotope Probing
Nature Protocols, 2007Co-Authors: Andrew S. Whiteley, Bruce C. Thomson, Tillmann Lueders, Mike ManefieldAbstract:Stable-Isotope Probing is a method used in microbial ecology that provides a means by which specific functional groups of organisms that incorporate particular substrates are identified without the prerequisite of cultivation. Stable-Isotope-labeled carbon (13C) or nitrogen (15N) sources are assimilated into microbial biomass of environmental samples. Separation and molecular analysis of labeled nucleic acids (DNA or RNA) reveals phylogenetic and functional information about the microorganisms responsible for the metabolism of a particular substrate. Here, we highlight general guidelines for incubating environmental samples with labeled substrate and provide a detailed protocol for separating labeled DNA from unlabeled community DNA. The protocol includes a modification of existing published methods, which maximizes the recovery of labeled DNA from CsCl gradients. The separation of DNA and retrieval of unlabeled and labeled fractions can be performed in 4-5 days, with much of the time being committed to the ultracentrifugation step.
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rna based stable isotope Probing and isolation of anaerobic benzene degrading bacteria from gasoline contaminated groundwater
Applied and Environmental Microbiology, 2006Co-Authors: Yuki Kasai, Mike Manefield, Yoh Takahata, Kazuya WatanabeAbstract:Stable isotope Probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [13C6]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the 13C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [14C]benzene showed that the labeled carbon was mostly incorporated into 14CO2 within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.
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enhanced sensitivity of dna and rrna based stable isotope Probing by fractionation and quantitative analysis of isopycnic centrifugation gradients
Environmental Microbiology, 2003Co-Authors: Tillmann Lueders, Mike Manefield, Michael W FriedrichAbstract:Stable isotope Probing (SIP) of nucleic acids allows the detection and identification of active members of natural microbial populations that are involved in the assimilation of an isotopically labelled compound into nucleic acids. SIP is based on the separation of isotopically labelled DNA or rRNA by isopycnic density gradient centrifugation. We have developed a highly sensitive protocol for the detection of 'light' and 'heavy' nucleic acids in fractions of centrifugation gradients. It involves the fluorometric quantification of total DNA or rRNA, and the quantification of either 16S rRNA genes or 16S rRNA in gradient fractions by real-time PCR with domain-specific primers. Using this approach, we found that fully 13C-labelled DNA or rRNA of Methylobacterium extorquens was quantitatively resolved from unlabelled DNA or rRNA of Methanosarcina barkeri by cesium chloride or cesium trifluoroacetate density gradient centrifugation respectively. However, a constant low background of unspecific nucleic acids was detected in all DNA or rRNA gradient fractions, which is important for the interpretation of environmental SIP results. Consequently, quantitative analysis of gradient fractions provides a higher precision and finer resolution for retrieval of isotopically enriched nucleic acids than possible using ethidium bromide or gradient fractionation combined with fingerprinting analyses. This is a prerequisite for the fine-scale tracing of microbial populations metabolizing 13C-labelled compounds in natural ecosystems.