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Journal ArticleDOI

Methane production from protozoan endosymbionts following stimulation of microbial metabolism within subsurface sediments

TL;DR: Results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.
Abstract: Previous studies have suggested that protozoa prey on Fe(III)- and sulfate-reducing bacteria that are enriched when acetate is added to uranium contaminated subsurface sediments to stimulate U(VI) reduction. In order to determine whether protozoa continue to impact subsurface biogeochemistry after these acetate amendments have stopped, 18S rRNA and s-tubulin sequences from this phase of an in situ uranium bioremediation field experiment were analyzed. Sequences most similar to Metopus species predominated, with the majority of sequences most closely related to M. palaeformis, a cilitated protozoan known to harbor methanogenic symbionts. Quantification of mcrA mRNA transcripts in the groundwater suggested that methanogens closely related to Metopus endosymbionts were metabolically active at this time. There was a strong correlation between the number of mcrA transcripts from the putative endosymbiotic methanogen and Metopus s-tubulin mRNA transcripts during the course of the field experiment, suggesting that the activity of the methanogens was dependent upon the activity of the Metopus species. Addition of the eukaryotic inhibitors cyclohexamide and colchicine to laboratory incubations of acetate-amended subsurface sediments significantly inhibited methane production and there was a direct correlation between methane concentration and Metopus s-tubulin and putative symbiont mcrA gene copies. These results suggest that, following the stimulation of subsurface microbial growth with acetate, protozoa harboring methanogenic endosymbionts become important members of the microbial community, feeding on moribund biomass and producing methane.

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Citations
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Journal ArticleDOI
TL;DR: In this paper , granular activated carbon (GAC) immobilized with riboflavin was added to an anaerobic digester treating food waste to stimulate direct interspecies electron transfer between bacteria and methanogens.

7 citations

Journal ArticleDOI
23 Sep 2022-mLife
TL;DR: In this paper , the transcriptome of Geobacter metallireducens during DIET-based growth with G. sulfurreducens reducing fumarate was compared with growing with diverse Methanosarcina.
Abstract: Direct interspecies electron transfer (DIET) may be most important in methanogenic environments, but mechanistic studies of DIET to date have primarily focused on cocultures in which fumarate was the terminal electron acceptor. To better understand DIET with methanogens, the transcriptome of Geobacter metallireducens during DIET-based growth with G. sulfurreducens reducing fumarate was compared with G. metallireducens grown in coculture with diverse Methanosarcina. The transcriptome of G. metallireducens cocultured with G. sulfurreducens was significantly different from those with Methanosarcina. Furthermore, the transcriptome of G. metallireducens grown with Methanosarcina barkeri, which lacks outer-surface c-type cytochromes, differed from those of G. metallireducens cocultured with M. acetivorans or M. subterranea, which have an outer-surface c-type cytochrome that serves as an electrical connect for DIET. Differences in G. metallireducens expression patterns for genes involved in extracellular electron transfer were particularly notable. Cocultures with c-type cytochrome deletion mutant strains, ∆Gmet_0930, ∆Gmet_0557 and ∆Gmet_2896, never became established with G. sulfurreducens but adapted to grow with all three Methanosarcina. Two porin–cytochrome complexes, PccF and PccG, were important for DIET; however, PccG was more important for growth with Methanosarcina. Unlike cocultures with G. sulfurreducens and M. acetivorans, electrically conductive pili were not needed for growth with M. barkeri. Shewanella oneidensis, another electroactive microbe with abundant outer-surface c-type cytochromes, did not grow via DIET. The results demonstrate that the presence of outer-surface c-type cytochromes does not necessarily confer the capacity for DIET and emphasize the impact of the electron-accepting partner on the physiology of the electron-donating DIET partner.

7 citations

Posted ContentDOI
12 Oct 2017-bioRxiv
TL;DR: It is demonstrated that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species and suggested that Methosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.
Abstract: Previous studies of in situ bioremediation of uranium-contaminated groundwater with acetate injections have focused on the role of Geobacter species in U(VI) reduction because of a lack of other abundant known U(VI)-reducing microorganisms. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40 days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. An enrichment culture dominated by a Methanosarcina species with the same Methanosarcina mcrA sequence that predominated in the field experiment could effectively convert acetate to methane. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of M. barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells, however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.

4 citations

DissertationDOI
01 Jan 2018
TL;DR: The aim of this thesis was to elucidate how various microbial communities work, with a focus on next generation sequencing data, and to discuss the relevant issues with modelling microbial communities, as well as overall scientific integrity in relationship with microbiome research.
Abstract: The aim of this thesis was to elucidate how various microbial communities work, with a focus on next generation sequencing data. The introduction in chapter 1 focuses on the history of biology, how the field of systems biology arose, and how the rise of nucleic acid sequencing has shaped a completely new field (among others), the microbiome research. In chapter 2, an overview is given how the microbiota can be studied, in connection to metabolic syndrome and its sub-pathologies, including obesity, type II diabetes, elevated blood pressure, and dyslipidemia. We summarize which different methodologies (16S rRNA amplicon sequencing, metagenomics, metatranscriptomics) can be used to investigate the microbiome with different foci, and how as a next step the microbiome can be modelled, in vitro and in silico. Chapter 3 describes the genome and transcriptome of the rat gut commensal Romboutsia ilealis CRIBT. We characterized genomic properties, including those related to metabolism and sporulation abilities. The transcriptome allowed us to investigate the organism’s carbohydrate degradation abilities, including its potential regulation. Chapter 4 is an investigation of an in vitro fermentation system, inoculated with human faecal material and the potential prebiotic Isomalto/malto-polysaccharides. The metatranscriptome of this system gave an insight into which genes are involved in the carbohydrate degradation, and which different types of organisms are involved and potentially need to cooperate for a full utilization of this carbohydrate. In chapter 5, the cow rumen microbiota is investigated under different feeding regimes. The metatranscriptome of the cow rumen microbiota showed distinct patterns depending on the ratio of starch or cellulose enriched feed components, namely maize vs. grass silage. The increase in starch led to a decrease in methane emissions of the cow rumen microbiota, which was reflected in the metatranscriptomics data. Most notably, lower expression levels of genes encoding for proteins involved in methanogenic pathways of the rumen archaeon Methanobrevibacter smithii was observed. The last chapter, the general discussion, mainly discusses the research described in this thesis with a focus on the relevant issues with modelling microbial communities, as well as overall scientific integrity in relationship with microbiome research.

4 citations


Cites background from "Methane production from protozoan e..."

  • ...The methanogenic Archaea were not directly affected by the diet, however, for methanogenesis they rely on metabolites produced by other community members (presumably a member of the Clostridiales in this case), which were decreased due to 15 the change in diet....

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  • ...directly affected, and this has been observed before in a different setting with intracellular Archaea [465]....

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Posted ContentDOI
03 Mar 2018-bioRxiv
TL;DR: Investigation of the impact of corn silage enhanced diets on the rumen microbiota of rumen-fistulated dairy cows and methanogenesis provided insights into key underlying mechanisms and opens the way for new rational methods to further reduce methane output of ruminants.
Abstract: Methane eructed by ruminant animals is a main contributor to greenhouse gas emissions and is solely produced by members of the phylum Euryarchaeota within the domain Archaea. Methanogenesis depends on the availability of hydrogen, carbon dioxide, methanol and acetate produced, which are metabolic products of anaerobic microbial degradation of feed-derived fibers. Changing the feed composition of the ruminants has been proposed as a strategy to mitigate methanogenesis of the rumen microbiota. We investigated the impact of corn silage enhanced diets on the rumen microbiota of rumen-fistulated dairy cows, with a special focus on carbohydrate breakdown and methanogenesis. Metatranscriptome analysis of rumen samples taken from animals fed corn silage enhanced diets revealed that genes involved in starch metabolism were significantly more expressed while archaeal genes involved in methanogenesis showed lower expression values. The nutritional intervention also influenced the cross-feeding between Archaea and Bacteria. The results indicate that the ruminant diet is important in methanogenesis. The diet-induced changes resulted in a reduced methane emission. The metatranscriptomic analysis provided insights into key underlying mechanisms and opens the way for new rational methods to further reduce methane output of ruminant animals.

2 citations


Cites background from "Methane production from protozoan e..."

  • ...General cellular processes, like replication, in which the cytoskeleton is involved, will then probably be directly affected, and this has been observed before in a different setting with intracellular Archaea (Holmes et al., 2014)....

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  • ...Closer inspection revealed that with an increase of CS in the diet, nearly all genes of the methanogenesis pathways were downregulated in a subset of the Archaea (expression profile C)....

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  • ...These groups included 13 genera (Bacteroides, Butyrivibrio, Clostridium, Entamoeba, Entodinium, Eubacterium, Faecalibacterium, Fibrobacter, Methanobrevibacter, Methanosphaera, Plasmodium, Prevotella, Ruminococcus) and 11 sequence clusters (not including the data assigned to the 13 genera) that could only be assigned at higher taxonomic levels (Archaea, Bacteria, Bacteroidales, Bacteroidetes, Clostridia, Clostridiales, Coriobacteriaceae, Eukaryota, Firmicutes, Methanobacteriaceae, Peptostreptococcaceae)....

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  • ...Furthermore one of these modules was found in the Archaea, which are not known to harbour either cellulosomal complexes or their starch counterparts....

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  • ...As several Archaea are endosymbionts of Protozoa, it can be speculated that an experimental change, which has an impact on the symbionts, will also affect their host (Finlay et al., 1994) (although this relationship is also not entirely clear (Morgavi et al., 2012))....

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References
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Journal ArticleDOI
TL;DR: A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original.
Abstract: The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.

70,111 citations

Journal ArticleDOI
TL;DR: ClUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W, providing an integrated system for performing multiple sequence and profile alignments and analysing the results.
Abstract: CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.

38,522 citations


"Methane production from protozoan e..." refers methods in this paper

  • ...Alignments were made in ClustalX (Thompson et al., 1997) and corrected with ProSeq v2....

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Journal ArticleDOI
TL;DR: An advanced version of the Molecular Evolutionary Genetics Analysis software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis, is released, which enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny.
Abstract: We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge.

37,956 citations


"Methane production from protozoan e..." refers methods in this paper

  • ...9 (Filatov, 2002) before phylogenetic trees were constructed with Mega v6 (Tamura et al., 2013)....

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01 Jan 2013
TL;DR: The Molecular Evolutionary Genetics Analysis (MEGA) software as discussed by the authors provides facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis, including the inference of timetrees.
Abstract: We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www. megasoftware.net free of charge.

30,478 citations

Journal ArticleDOI
TL;DR: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency.
Abstract: Background: Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a reader’s ability to evaluate critically the quality of the results presented or to repeat the experiments. Content: The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. Summary: Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

12,469 citations


"Methane production from protozoan e..." refers methods in this paper

  • ...Transcript abundances and qPCR efficiencies (90–99%) were calculated from appropriate standard curves and all qPCR experiments followed MIQE guidelines (Bustin et al., 2009)....

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  • ...%) were calculated from appropriate standard curves and all qPCR experiments followed MIQE guidelines (Bustin et al., 2009)....

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