RSS 1.0Imaging and Characterization Core Lab
Recent Submissions
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Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization.(Journal of nanobiotechnology, Springer Science and Business Media LLC, 2023-03-25) [Article]Background: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 μm and a width of 30–250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. Results: We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol–maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin–biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. Conclusion: The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion.
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Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens(Nature Communications, Springer Science and Business Media LLC, 2023-03-16) [Article]Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.
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Maximizing Active Fe Species in ZSM-5 Zeolite Using Organic-Template-Free Synthesis for Efficient Selective Methane Oxidation(Journal of the American Chemical Society, American Chemical Society (ACS), 2023-02-14) [Article]The selective oxidation of CH4 in the aqueous phase to produce valuable chemicals has attracted considerable attention due to its mild reaction conditions and simple process. As the most widely studied catalyst for this reaction, Fe-ZSM-5 demonstrates high intrinsic activity and selectivity; however, Fe-ZSM-5 prepared using conventional methods has a limited number of active Fe sites, resulting in low CH4 conversion per unit mass of the catalyst. This study reports a facile organic-template-free synthesis strategy that enables the incorporation of more Fe into the zeolite framework with a higher dispersion degree compared to conventional synthesis methods. Because framework Fe incorporated in this way is more readily transformed into isolated extra-framework Fe species under thermal treatment, the overall effect is that Fe-ZSM-5 prepared using this method (Fe-HZ5-TF) has 3 times as many catalytically active sites as conventional Fe-ZSM-5. When used for the selective oxidation of CH4 with 0.5 M H2O2 at 75 °C, Fe-HZ5-TF produced a record-high C1 oxygenate yield of 109.4 mmol gcat-1 h-1 (a HCOOH selectivity of 91.1%), surpassing other catalysts reported to date. Spectroscopic characterization and density functional theory calculations revealed that the active sites in Fe-HZ5-TF are mononuclear Fe species in the form of [(H2O)3Fe(IV)═O]2+ bound to Al pairs in the zeolite framework. This differs from conventional Fe-ZSM-5, where binuclear Fe acts as the active site. Analysis of the catalyst and product evolution during the reaction suggests a radical-driven pathway to explain CH4 activation at the mononuclear Fe site and subsequent conversion to C1 oxygenates.
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Highly Selective Photoelectroreduction of Carbon Dioxide to Ethanol over Graphene/Silicon Carbide Composites.(Angewandte Chemie (International ed. in English), Wiley, 2023-02-14) [Article]Using sunlight to produce valuable chemicals and fuels from carbon dioxide (CO2), i.e., artificial photosynthesis (AP) is a promising strategy to achieve solar energy storage and a negative carbon cycle. However, selective synthesis of C2 compounds with a high CO2 conversion rate remains challenging for current AP technologies. We performed CO2 photoelectroreduction over a graphene/silicon carbide (SiC) catalyst under simulated solar irradiation with ethanol (C2H5OH) selectivity of > 99% and a CO2 conversion rate of up to 17.1 mmol∙gcat-1·h-1 with sustained performance. Experimental and theoretical investigations indicated an optimal interfacial layer to facilitate the transfer of photogenerated electrons from the SiC substrate to the few-layer graphene overlayer, which also favored an efficient CO2 to C2H5OH conversion pathway.
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One-year outdoor operation of monolithic perovskite/silicon tandem solar cells(Cell Reports Physical Science, Elsevier BV, 2023-02-06) [Article]Perovskite/silicon tandem solar cells have gained significant attention as a viable commercial solution for ultra-high-efficiency photovoltaics. Ongoing research efforts focus on improving device performance, stability, and upscaling. Yet, paradoxically, their outdoor behavior remains largely unexplored. Here, we describe their performance over a complete calendar year outdoors in the area of the Red Sea coast of Saudi Arabia, which represents a hot and humid environment. After 1 year, our test device retains 80% of its initial power conversion efficiency. Further, we find three critical factors affecting current matching: the module temperature; deviations of the local, actual solar spectrum from the AM1.5G standard, which dictates optical design requirements of the subcells; and module soiling due to a spectrally non-uniform transmission of light through the accumulated dust. Overall, our results underline the promise of perovskite/silicon tandem solar cells as a future high-performance technology, yet device tailoring toward targeted deployment may be desired to achieve maximum energy yields.
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Elucidation of the alternating copolymerization mechanism of epoxides or aziridines with cyclic anhydrides in the presence of halide salts(Angewandte Chemie, Wiley, 2023-02-03) [Article]Organic halide salts in combination with metal or Lewis acids are the most common and essential catalysts in ring-opening copolymerizations (ROCOP). However, the role of organic halide salts was neglected. Here, we have uncovered the complex behavior of organic halide in ROCOP of epoxides or aziridines with cyclic anhydrides. Coordination of the chain-end cations, electron-withdrawing effect, leaving ability of halide atoms, chain-end basicity/nucleophilicity, and terminal steric hindrance cause three types of side reactions: single-site transesterification, substitution, and elimination. Understanding the complex functions of organic halide salts in ROCOP led us to develop highly active and selective aminocyclopropenium chlorides as catalysts/initiators. Adjustable H-bonding interactions of aminocyclopropenium with propagating anions and epoxides create chain-end coordination processes that generate highly reactive carboxylate and highly selective alkoxide chain ends.
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Formic Acid Dehydrogenation via an Active Ruthenium Pincer Catalyst Immobilized on Tetra-Coordinated Aluminum Hydride Species Supported on Fibrous Silica Nanospheres(ACS Catalysis, American Chemical Society (ACS), 2022-11-08) [Article]The demand for harmless and efficient energy sources is remarkably expanding, particularly after the increased awareness of global warming, greenhouse gas emissions, immense fossil fuel consumption, and so forth. Formic acid is considered a potential candidate as an energy carrier for reversible hydrogen storage owing to its decomposition to hydrogen (H2) and carbon dioxide (CO2) in the presence of suitable catalysts. However, selective and efficient decomposition of formic acid using classical heterogeneous catalysis is still challenging because most heterogeneous catalysts which are known are ill defined. Herein, we report a promising heterogeneous approach toward formic acid dehydrogenation using a ruthenium PN3P pincer complex, [Ru–H(CO) (tBuPN3P)] (I), immobilized on a fibrous silica nanosphere, KCC-1, with a strong Lewis acid character [(≡Si–O–Si≡) (≡Si–O−)2Al–H]. The resulting heterogeneous catalyst, [Ru(H) (CO) (tBuPN3P)]@[(≡Si–O–Si≡) (≡Si–O−)2Al–H] (III), has been fully characterized by advanced solid-state characterization techniques. In this compound, Al is tetrahedrally coordinated. It is a single-site catalyst which exhibits good stability toward water, high pressures, and high temperatures as well as good activity in formic acid dehydrogenation. An excellent turnover number of 600,000 and a recyclability of up to 45 cycles are observed.
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Ultra-Efficient Optical Gain and Lasing in MDACl2-Doped Perovskite Thin Films(Chemistry of Materials, American Chemical Society (ACS), 2022-10-25) [Article]Solution-processed perovskite materials have been enticing candidates for optical gain and lasing media because of their low cost, remarkable color purity, facile bandgap tunability, and high absorption cross-section. However, it is difficult, if not impossible, for them to highly amplify light with stable operations because they experience severe non-radiative emission losses due to their high density of surface and bulk defect centers and irregular composition. Here, we report that incorporating 5% of methylenediammonium dichloride (MDACl2) into mixed perovskite systems leads to a drastic reduction in the density of iodide interstitials and surface recombination losses. Subsequently, we record that MDA-treated, cavity-free thin perovskite films exhibit very photostable and ultra-low threshold amplified spontaneous emission of <6 μJ/cm2, approximately 30 times lower than the untreated films. Moreover, an ultra-high optical gain of 1000 cm–1 is successfully achieved, representing the highest reported gain for cavity-free perovskite films. These results are fully supported by extensive high-level density functional theory calculations and ultrafast transient absorption measurements. These findings will serve as a benchmark for the design and fabrication of low-threshold, high amplification perovskite media for lasers and light emission applications.
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Are hierarchical zeolites good catalysts for Methane Dehydroaromatization? A critical analysis(Catalysis Today, Elsevier BV, 2022-10-14) [Article]The socioeconomic dependence on natural gas must be reduced to comply with the stricter carbon emissions requirements. However, avoiding natural gas from the future energy mix is easier said than done. To bridge this gap, lignocellulosic biomass-derived biomethane (aka. “renewable natural gas”) represents an elegant solution to this contemporary problem. Owing to the increased industrial interest in hierarchically structured zeolites for biomethane valorization, through this work, we explore the technical feasibility and challenges associated with methane dehydroaromatization over Mo-loaded on both microporous and hierarchical zeolite ZSM-5. Hierarchical zeolites were prepared using inexpensive and environmentally benign glucose as a secondary organic structure directing agent, leading to two shapes (coffins and hexagonal bars) with comparable physicochemical properties. Although a similar catalytic performance was obtained over (nano-sized) microporous and hexagonal bar-derived zeolites, coffin-shaped zeolite catalysts led to lower activity and slower deactivation. Herein, catalyst deactivation was governed by inter- and intra-particle diffusional properties.
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Three-dimensional hierarchically porous MoS2 foam as high-rate and stable lithium-ion battery anode(Nature Communications, Springer Science and Business Media LLC, 2022-10-12) [Article]Architected materials that actively respond to external stimuli hold tantalizing prospects for applications in energy storage, wearable electronics, and bioengineering. Molybdenum disulfide, an excellent two-dimensional building block, is a promising candidate for lithium-ion battery anode. However, the stacked and brittle two-dimensional layered structure limits its rate capability and electrochemical stability. Here we report the dewetting-induced manufacturing of two-dimensional molybdenum disulfide nanosheets into a three-dimensional foam with a structural hierarchy across seven orders of magnitude. Our molybdenum disulfide foam provides an interpenetrating network for efficient charge transport, rapid ion diffusion, and mechanically resilient and chemically stable support for electrochemical reactions. These features induce a pseudocapacitive energy storage mechanism involving molybdenum redox reactions, confirmed by in-situ X-ray absorption near edge structure. The extraordinary electrochemical performance of molybdenum disulfide foam outperforms most reported molybdenum disulfide-based Lithium-ion battery anodes and state-of-the-art materials. This work opens promising inroads for various applications where special properties arise from hierarchical architecture.
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Cryo-electron structures of the extreme thermostable enzymes Sulfur Oxygenase Reductase and Lumazine Synthase(PLOS ONE, Public Library of Science (PLoS), 2022-10-03) [Article]Thermostable enzymes have the potential for use in a wide variety of biotechnological applications. Cryo-electron microscopy (cryo-EM) enables the imaging of biomolecules in their native aqueous environment. Here, we present high resolution cryo-EM structures of two thermostable enzymes that exhibit multimeric cage-like structures arranged into two different point-group symmetries. First, we determined the structure of the Sulfur Oxygenase Reductase (SOR) enzyme that catalyzes both the oxygenation and disproportionation of elemental sulfur in Archea and is composed of 24 homomeric units each of MW ≃ 35 kDa arranged in octahedral symmetry. The structure of SOR from Acidianus ambivalens (7X9W) was determined at 2.78 Å resolution. The active site of each subunit inside the central nanocompartment is composed of Fe3+ coordinated to two water molecules and the three amino acids (H86, H90 and E114). Second, we determined the structure of Lumazine Synthase (LS) from Aquifex aeolicus (7X7M) at 2.33 Å resolution. LS forms a cage-like structure consisting of 60 identical subunits each of MW ≃ 15 kDa arranged in a strict icosahedral symmetry. The LS subunits are interconnected by ion-pair network. Due to their thermostability and relatively easy purification scheme, both SOR and LS can serve as a model for the catalytic and structural characterization of biocatalysts as well as a benchmark for cryo-EM sample preparation, optimization of the acquisition parameters and 3D reconstruction.
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Potential-induced degradation in perovskite/silicon tandem photovoltaic modules(Cell Reports Physical Science, Elsevier BV, 2022-08-31) [Article]Despite great progress in perovskite/silicon tandem solar cells’ device performance, their susceptibility to potential-induced degradation (PID) remains unexplored. In this study, we find that applying a voltage bias of −1,000 V to single-device perovskite/silicon tandem modules at 60°C for ∼1 day can cause a ∼50% loss in their power conversion efficiency, which raises concerns for tandem commercialization. We found no accumulation of Na+ in the perovskite or silicon photon absorbers. Consequently, no obvious shunt is observed in our silicon subcells. We also find that elements diffuse from the perovskite into the module encapsulant during PID testing. We argue that this diffusion is the main PID mechanism in our tandem modules. While applying a large positive voltage bias can partially recover this PID, introducing barriers or structures to prevent elemental diffusion out of the perovskite may be required to mitigate this degradation phenomenon.
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Illustrating the overall reaction network of the synthesis-gas-to-hydrocarbons process over iron-zeolite bifunctional catalysis(Chem Catalysis, Elsevier BV, 2022-08-24) [Article]Since its discovery in the early 20th century, Fischer-Tropsch synthesis (FTS) has opened a path, as an alternative to crude oil, to produce fuels and chemicals. When classical FTS catalysts are combined with acidic zeolites, the scope of this gas-phase polymerization can be narrowed, thus maximizing the production of value-added commodities and eliminating energy-consuming separation steps. However, from a mechanistic standpoint, even now, little is known about the role of the different reaction intermediates. Here, we present a comprehensive, in-depth, mechanistic investigation using solid-state NMR spectroscopy and well-designed control experiments on combining a classical Fe-based FTS catalyst and zeolites with different topologies to establish the impact of “co-catalytic” key organic carbon-based reaction intermediates, including carbonylated/oxygenated species (ester/ketone/alcohol/ether/epoxide/ketene). Consequently, this work provides experimental evidence supporting the “co-existence” of oxygenate (cf. surface-enol and CO-insertion) mechanisms (together with the traditional carbide-based FTS mechanism). The significance of “supramolecular reactive centers” within zeolite and host-guest chemistry has also been illuminated.
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Structural evolution and strain generation of derived-Cu catalysts during CO2 electroreduction(Nature Communications, Springer Science and Business Media LLC, 2022-08-18) [Article]Copper (Cu)-based catalysts generally exhibit high C2+ selectivity during the electrochemical CO2 reduction reaction (CO2RR). However, the origin of this selectivity and the influence of catalyst precursors on it are not fully understood. We combine operando X-ray diffraction and operando Raman spectroscopy to monitor the structural and compositional evolution of three Cu precursors during the CO2RR. The results indicate that despite different kinetics, all three precursors are completely reduced to Cu(0) with similar grain sizes (~11 nm), and that oxidized Cu species are not involved in the CO2RR. Furthermore, Cu(OH)2- and Cu2(OH)2CO3-derived Cu exhibit considerable tensile strain (0.43%~0.55%), whereas CuO-derived Cu does not. Theoretical calculations suggest that the tensile strain in Cu lattice is conducive to promoting CO2RR, which is consistent with experimental observations. The high CO2RR performance of some derived Cu catalysts is attributed to the combined effect of the small grain size and lattice strain, both originating from the in situ electroreduction of precursors. These findings establish correlations between Cu precursors, lattice strains, and catalytic behaviors, demonstrating the unique ability of operando characterization in studying electrochemical processes.
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Temperature-Dependent Optical Modeling of Perovskite Solar Cells(The Journal of Physical Chemistry C, American Chemical Society (ACS), 2022-08-12) [Article]Comprehensive temperature-dependent optical modeling of perovskite solar cells (PSCs) and modules is essential to accurately predict their energy yield and quantify their energy losses under real-world operating conditions, where devices are subject to different irradiance spectra and intensities as well as operating temperatures. These models require the accurate determination of the temperature-dependent optical constants of perovskites. Here, we report on these data, empirically determined via spectroscopic ellipsometry, for triple-cation perovskites with band gaps ranging between 1.58 and 1.77 eV at temperatures between 25 and 75 °C. Using this data set, we develop a simple empirical model to obtain the temperature-dependent optical constants of perovskites of an arbitrary band gap. We validate our empirical model by comparing the measured temperature-dependent short-circuit current densities and external quantum efficiency data of single-junction PSCs with simulated results using the modeled optical constants.
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Topochemical Synthesis of Ca3CrN3H Involving a Rotational Structural Transformation for Catalytic Ammonia Synthesis(Angewandte Chemie (International ed. in English), Wiley, 2022-08-05) [Article]Topochemical reactions have led to great progress in the discovery of new metastable compounds with novel chemical and physical properties. With these reactions, the overall crystal structure of the host material is generally maintained. Here we report a topochemical synthesis of a hexagonal nitride hydride, h-Ca3CrN3H, by heating an orthorhombic nitride, o-Ca3CrN3, under hydrogen at 673 K, accompanied by a rotational structural transformation. The hydrogen intercalation modifies the Ca-N rock-salt-like atomic packing in o-Ca3CrN3 to a face-sharing octahedral chain in h-Ca3CrN3H, mimicking a 'hinged tessellation' movement. In addition, the h-Ca3CrN3H exhibited stable ammonia synthesis activity when used as a catalyst.
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Understanding catalyst deactivation during the direct cracking of crude oil(CATALYSIS SCIENCE & TECHNOLOGY, Royal Society of Chemistry (RSC), 2022-08-03) [Article]The increasing demand for base chemicals i.e., ethylene and propylene, along with the expected peak in gasoline and fuels demand, are stirring intense research into refineries to be built around processes that maximize the production of chemicals (oil to chemicals, OTC, processes). One of the main challenges at hand for OTC technologies is the formulation of appropriate catalysts able to handle the wide boiling point of the feed and to withstand continuous operation at industrial scale. Hydrothermal degradation, coke deposition and the presence of impurities, such as metals, sulfur and nitrogen containing species, in the feedstock affect catalyst lifetime, activity and selectivity. In this work, we evaluate long term catalyst stability along with the main causes of reversible and irreversible catalyst deactivation. Our results demonstrate that formulation prevents, to a large extent, the degradation of the zeolitic components of the catalyst. Metal deposition, on the other hand, results in a slight decrease in activity along with partial changes in selectivity patterns. The main reasons behind these changes are discussed in detail with the help of extensive characterization.
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The Gardenia Carotenoid Cleavage Dioxygenase 4a is an efficient tool for biotechnological production of crocins in green and non-green plant tissues(Plant Biotechnology Journal, Wiley, 2022-07-29) [Article]Crocins are beneficial antioxidants and potential chemotherapeutics that give raise, together with picrocrocin, to the color and taste of saffron, the most expensive spice, respectively. Crocins are formed from crocetin dialdehyde that is produced in Crocus sativus from zeaxanthin by the Carotenoid Cleavage Dioxygenase 2L (CsCCD2L), while GjCCD4a from Gardenia jasminoides, another major source of crocins, converted different carotenoids, including zeaxanthin, into crocetin dialdehyde in bacterio. To establish a biotechnological platform for sustainable production of crocins, we investigated the enzymatic activity of GjCCD4a, in comparison to CsCCD2L, in citrus callus engineered by Agrobacterium-mediated super-transformation of multi genes and in transiently transformed Nicotiana benthamiana leaves. We demonstrate that co-expression of GjCCD4a with phytoene synthase and β-carotene hydroxylase genes is an optimal combination for heterologous production of crocetin, crocins and picrocrocin in citrus callus. By profiling apocarotenoids and using in vitro assays, we show that GjCCD4a cleaved β-carotene, in planta, and produced crocetin dialdehyde via C30 β-apocarotenoid intermediate. GjCCD4a also cleaved C27 β-apocarotenoids, providing a new route for C17-dialdehyde biosynthesis. Callus lines overexpressing GjCCD4a contained higher number of plastoglobuli in chromoplast-like plastids and increased contents in phytoene, C17:0 fatty acid (FA), and C18:1 cis-9 and C22:0 FA esters. GjCCD4a showed a wider substrate specificity and higher efficiency in Nicotiana leaves, leading to the accumulation of up to 1.6 mg/g dry weight crocins. In summary, we established a system for investigating CCD enzymatic activity in planta and an efficient biotechnological platform for crocins production in green and non-green crop tissues/organs.
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Asymmetric pore windows in MOF membranes for natural gas valorization(Nature, Springer Science and Business Media LLC, 2022-06-22) [Article]To use natural gas as a feedstock alternative to coal and oil, its main constituent, methane, needs to be isolated with high purity1. In particular, nitrogen dilutes the heating value of natural gas and is, therefore, of prime importance for removal2. However, the inertness of nitrogen and its similarities to methane in terms of kinetic size, polarizability and boiling point pose particular challenges for the development of energy-efficient nitrogen-removing processes3. Here we report a mixed-linker metal–organic framework (MOF) membrane based on fumarate (fum) and mesaconate (mes) linkers, Zr-fum67-mes33-fcu-MOF, with a pore aperture shape specific for effective nitrogen removal from natural gas. The deliberate introduction of asymmetry in the parent trefoil-shaped pore aperture induces a shape irregularity, blocking the transport of tetrahedral methane while allowing linear nitrogen to permeate. Zr-fum67-mes33-fcu-MOF membranes exhibit record-high nitrogen/methane selectivity and nitrogen permeance under practical pressures up to 50 bar, removing both carbon dioxide and nitrogen from natural gas. Techno-economic analysis shows that our membranes offer the potential to reduce methane purification costs by about 66% for nitrogen rejection and about 73% for simultaneous removal of carbon dioxide and nitrogen, relative to cryogenic distillation and amine-based carbon dioxide capture.
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Thicker Ice Improves the Integrity and Angular Distribution of CDC48A Hexamers on Cryo-EM Grids(Frontiers in Molecular Biosciences, Frontiers Media SA, 2022-06-17) [Article]Many cryogenic electron microscopy (cryo-EM) single particle analyses are constrained by the sample preparation step upon which aggregation, dissociation, and/or preferential orientation of particles can be introduced. Here, we report how we solved these problems in the case of CDC48A, a hexameric AAA ATPase from Arabidopsis thaliana. CDC48A hexamers are well preserved under negative staining conditions but disassemble during grid freezing using the classical blotting method. Vitrification of grids using the blot-free Chameleon method preserved the integrity of particles but resulted in their strong preferential orientation. We then used a strategy where we improved in parallel the purification of CDC48A and the conditions for cryo-EM data acquisition. Indeed, we noted that images taken from thicker ice presented an even distribution of intact particles with random orientations, but resulted in a lower image resolution. Consequently, in our case, distribution, orientation, image resolution, and the integrity of particles were tightly correlated with ice thickness. By combining the more homogeneous and stable CDC48A hexamers resulting from our improved purification protocol with an iterative search across different ice thicknesses, we identified an intermediate thickness that retained sufficiently high-resolution structural information while maintaining a complete distribution of particle orientations. Our approach may provide a simple, fast, and generally applicable strategy to record data of sufficient quality under standard laboratory and microscope settings. This method may be of particular value when time and resources are limited.
