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      Genome-wide transcriptional analysis suggests hydrogenase- and nitrogenase-mediated hydrogen production in Clostridium butyricum CWBI 1009

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          Abstract

          Background

          Molecular hydrogen, given its pollution-free combustion, has great potential to replace fossil fuels in future transportation and energy production. However, current industrial hydrogen production processes, such as steam reforming of methane, contribute significantly to the greenhouse effect. Therefore alternative methods, in particular the use of fermentative microorganisms, have attracted scientific interest in recent years. However the low overall yield obtained is a major challenge in biological H 2 production. Thus, a thorough and detailed understanding of the relationships between genome content, gene expression patterns, pathway utilisation and metabolite synthesis is required to optimise the yield of biohydrogen production pathways.

          Results

          In this study transcriptomic and proteomic analyses of the hydrogen-producing bacterium Clostridium butyricum CWBI 1009 were carried out to provide a biomolecular overview of the changes that occur when the metabolism shifts to H 2 production. The growth, H 2-production, and glucose-fermentation profiles were monitored in 20 L batch bioreactors under unregulated-pH and fixed-pH conditions (pH 7.3 and 5.2). Conspicuous differences were observed in the bioreactor performances and cellular metabolisms for all the tested metabolites, and they were pH dependent. During unregulated-pH glucose fermentation increased H 2 production was associated with concurrent strong up-regulation of the nitrogenase coding genes. However, no such concurrent up-regulation of the [FeFe] hydrogenase genes was observed. During the fixed pH 5.2 fermentation, by contrast, the expression levels for the [FeFe] hydrogenase coding genes were higher than during the unregulated-pH fermentation, while the nitrogenase transcripts were less abundant. The overall results suggest, for the first time, that environmental factors may determine whether H 2 production in C. butyricum CWBI 1009 is mediated by the hydrogenases and/or the nitrogenase.

          Conclusions

          This work, contributing to the field of dark fermentative hydrogen production, provides a multidisciplinary approach for the investigation of the processes involved in the molecular H 2 metabolism of clostridia. In addition, it lays the groundwork for further optimisation of biohydrogen production pathways based on genetic engineering techniques.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13068-015-0203-5) contains supplementary material, which is available to authorized users.

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          Most cited references36

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          Current two-dimensional electrophoresis technology for proteomics.

          Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular mass (Mr), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where Mr and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037-1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007-4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5-12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (delta pI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (approximately 2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2-DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two-dimensional protein databases.
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            Polyamines: essential factors for growth and survival.

            Polyamines are low molecular weight, aliphatic polycations found in the cells of all living organisms. Due to their positive charges, polyamines bind to macromolecules such as DNA, RNA, and proteins. They are involved in diverse processes, including regulation of gene expression, translation, cell proliferation, modulation of cell signalling, and membrane stabilization. They also modulate the activities of certain sets of ion channels. Because of these multifaceted functions, the homeostasis of polyamines is crucial and is ensured through regulation of biosynthesis, catabolism, and transport. Through isolation of the genes involved in plant polyamine biosynthesis and loss-of-function experiments on the corresponding genes, their essentiality for growth is reconfirmed. Polyamines are also involved in stress responses and diseases in plants, indicating their importance for plant survival. This review summarizes the recent advances in polyamine research in the field of plant science compared with the knowledge obtained in microorganisms and animal systems.
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              Biohydrogen production: prospects and limitations to practical application

              D. Levin (2004)
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                Author and article information

                Contributors
                magdalena.calusinska@list.lu
                hamiltonbolton@gmail.com
                pmonsieu@sckcen.be
                gregory.mathy@ucb.com
                nleys@sckcen.be
                f.franck@ulg.ac.be
                bjoris@ulg.ac.be
                p.thonart@ulg.ac.be
                s.hiligsmann@ulg.ac.be
                awilmotte@ulg.ac.be
                Journal
                Biotechnol Biofuels
                Biotechnol Biofuels
                Biotechnology for Biofuels
                BioMed Central (London )
                1754-6834
                22 February 2015
                22 February 2015
                2015
                : 8
                : 27
                Affiliations
                [ ]Centre for Protein Engineering, Bacterial Physiology and Genetics, University of Liège, Allée de la Chimie 3, B-4000 Liège, Belgium
                [ ]Walloon Centre of Industrial Biology, University of Liège, Boulevard du Rectorat 29, B-4000 Liège, Belgium
                [ ]Microbiology Unit, Expertise Group for Molecular and Cellular Biology, Institute for Environment, Health and Safety, Belgian Nuclear Research Centre (SCK-CEN), Boeretang 200, B-2400 Mol, Belgium
                [ ]Bioenergetics Laboratory, University of Liège, Boulevard du Rectorat 27, B-4000 Liège, Belgium
                [ ]Environmental Research and Innovation Department, Luxembourg Institute of Science and Technology, Rue du Brill 41, L-4422 Belvaux, Luxembourg
                Article
                203
                10.1186/s13068-015-0203-5
                4342158
                25722742
                4bb5dd6c-3ccb-40c7-b56a-1233469915a5
                © Calusinska et al.; licensee BioMed Central. 2015

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 12 September 2014
                : 12 January 2015
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2015

                Biotechnology
                dark fermentation,clostridium butyricum,[fefe] hydrogenase,nitrogenase,rna-seq,2d-dige
                Biotechnology
                dark fermentation, clostridium butyricum, [fefe] hydrogenase, nitrogenase, rna-seq, 2d-dige

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