Project description:Glycerol is an attractive feedstock for biofuels since it accumulates as a byproduct during biodiesel operations; hence, it is interesting to consider converting glycerol to hydrogen using the formate hydrogen lyase system of Escherichia coli which converts pyruvate to hydrogen. Starting with Escherichia coli BW25113 frdC that lacks fumarate reductase to eliminate the negative effect of accumulated hydrogen on glycerol fermentation and by using both adaptive evolution and chemical mutagenesis combined with a selection method based on increased growth on glycerol, we obtained an improved strain, HW2, that produces 20-fold more hydrogen in glycerol medium (0.68 mmol/L/h) compared to that of frdC mutant. HW2 also grows 5-fold faster (0.25 1/h) than BW25113 frdC on glycerol, so it achieves a reasonable growth rate. Corroborating the increase in hydrogen production, glycerol dehydrogenase activity in HW2 increased 4-fold compared to BW25113 frdC. In addition, a whole-transcriptome study revealed that several pathways that would decrease hydrogen yields were repressed in HW2 (fbp, focA, and gatYZ) while a beneficial pathway, eno which encodes enolase was induced.
Project description:Metabolic flexibility in aerobic methane oxidising bacteria (methanotrophs) enhances cell growth and survival in instances where resources are variable or limiting. Examples include the production of intracellular compounds (such as glycogen or polyhydroxyalkanoates) in response to unbalanced growth conditions and the use of some energy substrates, besides methane, when available. Indeed, recent studies show that verrucomicrobial methanotrophs can grow mixotrophically through oxidation of hydrogen and methane gases via respiratory membrane-bound group 1d [NiFe] hydrogenases and methane monooxygenases respectively. Hydrogen metabolism is particularly important for adaptation to methane and oxygen limitation, suggesting this metabolic flexibility may confer growth and survival advantages. In this work, we provide evidence that, in adopting a mixotrophic growth strategy, the thermoacidophilic methanotroph, Methylacidiphilum sp. RTK17.1 changes its growth rate, biomass yields and the production of intracellular glycogen reservoirs. Under nitrogen-fixing conditions, removal of hydrogen from the feed-gas resulted in a 14 % reduction in observed growth rates and a 144% increase in cellular glycogen content. Concomitant with increases in glycogen content, the total protein content of biomass decreased following the removal of hydrogen. Transcriptome analysis of Methylacidiphilum sp. RTK17.1 revealed a 3.5-fold upregulation of the Group 1d [NiFe] hydrogenase in response to oxygen limitation and a 4-fold upregulation of nitrogenase encoding genes (nifHDKENX) in response to nitrogen limitation. Genes associated with glycogen synthesis and degradation were expressed constitutively and did not display evidence of transcriptional regulation. Collectively these data further challenge the belief that hydrogen metabolism in methanotrophic bacteria is primarily associated with energy conservation during nitrogen fixation and suggests its utilisation provides a competitive growth advantage within hypoxic habitats.
Project description:Anaerobic microorganisms producing hydrogen and methane from the co-digestion of glycerol and domestic wastewater in single-stage and two-stage anaerobic fluidized bed reactors under mesophilic conditions. Metagenome
Project description:Glycerol is an attractive feedstock for biofuels since it accumulates as a byproduct during biodiesel operations; hence, it is interesting to consider converting glycerol to hydrogen using the formate hydrogen lyase system of Escherichia coli which converts pyruvate to hydrogen. Starting with Escherichia coli BW25113 frdC that lacks fumarate reductase to eliminate the negative effect of accumulated hydrogen on glycerol fermentation and by using both adaptive evolution and chemical mutagenesis combined with a selection method based on increased growth on glycerol, we obtained an improved strain, HW2, that produces 20-fold more hydrogen in glycerol medium (0.68 mmol/L/h) compared to that of frdC mutant. HW2 also grows 5-fold faster (0.25 1/h) than BW25113 frdC on glycerol, so it achieves a reasonable growth rate. Corroborating the increase in hydrogen production, glycerol dehydrogenase activity in HW2 increased 4-fold compared to BW25113 frdC. In addition, a whole-transcriptome study revealed that several pathways that would decrease hydrogen yields were repressed in HW2 (fbp, focA, and gatYZ) while a beneficial pathway, eno which encodes enolase was induced. the overnight aerobic culture of HW2 and the frdC mutant in LB (25mL) were sparged with nitrogen for 5 min. Sealed crimp-top vials (60 mL) were also purged with nitrogen for 5 min. Inside an anaerobic glove-box, 30 mL of sparged uninoculated glycerol medium and 3 mL of sparged overnight culture were added to each vial, then the vials were kept at 37oC with shaking for 5 h for cell collection and RNA isolation.
Project description:Methanococcus maripaludis is a methanogenic Archaea that conserves energy from molecular hydrogen to reduce carbon dioxide to methane. Chemostat grown cultures limited for phosphate or leucine were compared to determine the regulatory response to leucine limitation. Keywords: archaea, hydrogen, leucine, phosphate, nutrient limitation, growth rate, methanogen
Project description:Methanococcus maripaludis is a methanogenic Archaea that conserves energy from molecular hydrogen to reduce carbon dioxide to methane. Chemostat grown cultures limited for hydrogen, phosphate, or leucine were compared to determine the regulatory response to hydrogen limitation. This was done by comparing hydrogen limited cultures to both leucine limited and phosphate limited cultures. Slow and rapid growing samples limited for either hydrogen or phosphate were compared to determine the regulatory effects of growth rate. Keywords: archaea, hydrogen, leucine, phosphate, nutrient limitation, growth rate, methanogen
Project description:Fermenting microbial communities generate hydrogen: its removal through production of acetate, methane, or hydrogen sulfide modulates the efficiency of energy extraction from available nutrients in many ecosystems. We noted that pathway components for acetogenesis are more abundantly and consistently represented in the gut microbiomes of monozygotic twins and their mothers than components for methanogenesis or sulfate reduction, and subsequently analyzed the metabolic potential of two sequenced human gut acetogens, Blautia hydrogenotrophica and Marvinbryantia formatexigens in vitro and in the intestines of gnotobiotic mice harboring a prominent saccharolytic bacterium. To do so, we developed a generally applicable method for multiplex sequencing of expressed microbial mRNAs, and together with mass spectrometry of metabolites, show that these organisms have distinct patterns of substrate utilization. B. hydrogenotrophica targets aliphatic and aromatic amino acids. It increases the efficiency of fermentation by consuming reducing equivalents, thereby maintaining a high NAD+/NADH ratio and boosting acetate production. In contrast, M. formatexigens consumes oligosaccharides, does not impact the redox state of the gut, and boosts the yield of succinate. These findings have strategic implications for those who wish to manipulate the hydrogen economy of gut microbial communities in ways that modulate energy harvest.