Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase

The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F_(420)-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H_2-CO_2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO2 in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H_2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech Δfrh and Δech Δfrh Δvht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H_2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO_2 produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F_(420) dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F_(420). The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed

MEA Manufacturing using an Additive Manufacturing Process to Deposit a Catalyst Pattern in an MEA and Its Impact on Cost Reduction

The manufacturing of a fuel cell Membrane Electrode Assembly (MEA) is a significant cost driver in polymer-electrolyte membrane (PEM) fuel cell technologies, primarily due to the inclusion of expensive materials in the catalyst layer. The selective deposition of a catalyst on the MEA of a fuel cell can drastically reduce the costs depending upon the catalyst, method used for deposition, and production volume. In this paper, testing and analysis of a novel catalyst iridium oxide is discussed. The performance of the catalyst will be compared with the conventional catalysts which will give us an estimate of its effectiveness however, in this paper, only its feasibility in terms of cost is discussed

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage PhiC31. Host strains expressing the PhiC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetRgene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion

Laser Based Rapid Manufacturing of Metallic Gas Diffusion Layers for PEM Fuel Cells

Gas Diffusion layers (GDL’s) are an essential component of Polymer Electrolyte Membrane Fuel cells (PEMFC’s) which aid in thermal & electrical conductivities, water management and act as backup layers for the membrane electrode assemblies. This paper summarizes the effort to prototype metallic GDL designs using a miniature laser deposition system developed at Missouri University of Science & Technology. The pore sizes are controlled by masking the diverging laser beam using stainless steel masks of varying sizes and shapes. The through pore feature and further treatment of the GDL’s for hydrophobicity reduces the water management issue and thereby increases the performance of the fuel cells. The operational characteristics of the GDL can be optimized by understanding the effect of the key parameters like fluid permeability, porosity, hydrophobicity and the surface morphology

Fuel Cell Development using Additive Manufacturing Technologies -- A Review

Fuel cells are being perceived as the future clean energy source by many developed countries in the world. The key today for clean power is the reliance of fuel cells not only to power automobiles but also for residential, small commercial, backup power etc. which calls for production on a large scale. Additive manufacturing is perceived as a way to develop cost effective fuel cells. It imparts flexibility to design different kinds of fuel cells along with reduction in material wastage. This paper deals with the review of additive manufacturing processes for research and development of fuel cell components, such as synthesizing and prototyping new materials for fuel cell components, fuel cell system design and prototyping, designing well sealed fuel cells, bridging from fuel cell design to manufacturing tooling, etc

Hopanoids Producing Bacteria and Related Biofertilizers, Compositions, Methods and Systems

Hopanoids, hopanoids-producing nitrogen-fixing bacteria, and related formulations, systems and methods are described herein. In particular, hopanoids alone or in combination with hopanoid-producing nitrogen-fixing bacteria can be used as biofertilizer to stimulate plant growth and yield with enhanced tolerance to diverse stresses found in plant-microbe symbiotic microenvironments

The General Stress Response Factor EcfG Regulates Expression of the C-2 Hopanoid Methylase HpnP in Rhodopseudomonas palustris TIE-1

Lipid molecules preserved in sedimentary rocks facilitate the reconstruction of events that have shaped the evolution of the Earth's biosphere. A key limitation for the interpretation of many of these molecular fossils is that their biological roles are still poorly understood. Here, we use Rhodopseudomonas palustris TIE-1 to identify factors that induce biosynthesis of 2-methyl hopanoids (2-MeBHPs), progenitors of 2-methyl hopanes, one of the most abundant biomarkers in the rock record. This is the first dissection of the regulation of hpnP, the gene encoding the C-2 hopanoid methylase, at the molecular level. We demonstrate that EcfG, the general stress response factor of alphaproteobacteria, regulates expression of hpnP under a variety of challenges, including high temperature, pH stress, and presence of nonionic osmolytes. Although higher hpnP transcription levels did not always result in higher amounts of total methylated hopanoids, the fraction of a particular kind of hopanoid, 2-methyl bacteriohopanetetrol, was consistently higher in the presence of most stressors in the wild type, but not in the ΔecfG mutant, supporting a beneficial role for 2-MeBHPs in stress tolerance. The ΔhpnP mutant, however, did not exhibit a growth defect under the stress conditions tested except in acidic medium. This indicates that the inability to make 2-MeBHPs under most of these conditions can readily be compensated. Although stress is necessary to regulate 2-MeBHP production, the specific conditions under which 2-MeBHP biosynthesis is essential remain to be determined

Energy Conservation via Hydrogen Cycling in the Methanogenic Archaeon Methanosarcina barkeri

Energy conservation via hydrogen cycling, which generates proton motive force by intracellular H_2 production coupled to extracellular consumption, has been controversial since it was first proposed in 1981. It was hypothesized that the methanogenic archaeon Methanosarcina barkeri is capable of energy conservation via H_2 cycling, based on genetic data that suggest that H_2 is a preferred, but nonessential, intermediate in the electron transport chain of this organism. Here, we characterize a series of hydrogenase mutants to provide direct evidence of H_2 cycling. M. barkeri produces H_2 during growth on methanol, a phenotype that is lost upon mutation of the cytoplasmic hydrogenase encoded by frhADGB, although low levels of H_2, attributable to the Ech hydrogenase, accumulate during stationary phase. In contrast, mutations that conditionally inactivate the extracellular Vht hydrogenase are lethal when expression of the vhtGACD operon is repressed. Under these conditions, H_2 accumulates, with concomitant cessation of methane production and subsequent cell lysis, suggesting that the inability to recapture extracellular H_2 is responsible for the lethal phenotype. Consistent with this interpretation, double mutants that lack both Vht and Frh are viable. Thus, when intracellular hydrogen production is abrogated, loss of extracellular H_2 consumption is no longer lethal. The common occurrence of both intracellular and extracellular hydrogenases in anaerobic microorganisms suggests that this unusual mechanism of energy conservation may be widespread in nature

Genetic, Biochemical, and Molecular Characterization of Methanosarcina barkeri Mutants Lacking Three Distinct Classes of Hydrogenase

The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F_(420)-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H_2-CO_2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO2 in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H_2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the Δech Δfrh and Δech Δfrh Δvht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H_2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO_2 produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F_(420) dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F_(420). The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed