Atomic force phase imaging for dynamic detection of adsorbed hydrogen on a catalytic palladium surface under liquid.

Dynamic observation of hydrogen on catalytic metal surfaces is a challenging aspect of studying liquid-phase heterogeneous catalysis. Current methods suffer from one or more of the following limitations: the requirement to observe the surface in high vacuum, the inability to provide nanometer-level spatial resolution, the inability to deal with opaque catalysts and/or liquid immersion phase, the lack of real-time scanning of the surface area, and the inability to assess pronounced topographies or mixed materials. Atomic force microscopy (AFM) phase-shift imaging remedies these issues and provides an opportunity for dynamic direct observation of catalyst surfaces at or near actual reaction conditions immersed in liquid. Hydrogen was delivered to a palladium surface immersed in water by diffusion through a support film of dense polycarbonate. The palladium surface was continuously probed by tapping-mode AFM. The theoretically predicted time-dependent appearance of hydrogen on the water-covered palladium surface matched the experimental observation reasonably well. The technique demonstrated here is unique in that the appearance of hydrogen is dynamically detected in real time on a catalyst surface immersed in water with nanometer-scale spatial resolution. The results presented here supply a new level of information for heterogeneous catalysis that is not available with existing techniques. This work opens new avenues in the study of heterogeneous catalysis, a field with tremendous practical importance and serious analytical challenges.

Evaluation of green solvents: Oil extraction from oleaginous yeast Lipomyces starkeyi using cyclopentyl methyl ether (CPME).

Cyclopentyl methyl ether (CPME) was evaluated for extracting oil or triacylglycerol (TAG) from wet cells of the oleaginous yeast Lipomyces starkeyi. CPME is a greener alternative to chloroform as a potential solvent for oil recovery. A monophasic system of CPME and biphasic system of CPME:water (1:0.7) performed poorly having the lowest TAG extraction efficiency and TAG selectivity compared to other monophasic systems of hexane and chloroform and the biphasic Bligh and Dyer method (chloroform:methanol:water). Biphasic systems of CPME:water:alcohol (methanol/ethanol/1-propanol) were tested and methanol achieved the best oil extraction efficiency compared to ethanol and 1-propanol. Different biphasic systems of CPME:methanol:water were tested, the best TAG extraction efficiency and TAG selectivity achieved was 9.9 mg/mL and 64.6%, respectively, using a starting ratio of 1:1.7:0.6 and a final ratio of 1:1:0.8 (CPME:methanol:water). Similar results were achieved for the Bligh and Dyer method (TAG extraction efficiency of 10.2 mg/mL and TAG selectivity of 66.0%) indicating that the biphasic CPME system was comparable. The fatty acid profile remained constant across all the solvent systems tested indicating that choice of solvent was not specific for any certain fatty acid. This study was able to demonstrate that CPME could be used as an alternative solvent for the extraction of oil from the wet biomass of oleaginous yeast. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1096-1103, 2017.

Oleaginous yeast: a value-added platform for renewable oils.

Yeast single cell oil (SCO) is a non-crop-based, renewable oil source that can be used for the production of bio-based oleochemicals. Stand-alone production of SCO for oleochemicals is currently not cost-competitive because lower-cost alternatives from petroleum and crop-based resources are available. Utilizing low-valued nutrient sources, implementing cost-efficient downstream processes and adopting biotechnological advancements such as systems biology and metabolic engineering could prove valuable in making an SCO platform a reality in the emerging bio-based economy. This review aims to consider key biochemical pathways for storage lipid synthesis, possible pathways for SCO yield improvement, previously used bioprocessing techniques for SCO production, challenges in SCO commercialization and advantages of adopting a renewable SCO platform.

Analysis of atomic force microscopy phase data to dynamically detect adsorbed hydrogen under ambient conditions.

Characterization of the interactions of hydrogen with catalytic metal surfaces and the mass transfer processes involved in heterogeneous catalysis are important for catalyst development. Although a range of technologies for studying catalytic surfaces exist, much of it relies on high-vacuum conditions that preclude in situ research. In contrast, atomic force microscopy (AFM) provides an opportunity for direct observation of surfaces under or near actual reaction conditions. Tapping-mode AFM was explored here because it expands AFM beyond the usual topographic information toward speciation and other more subtle surface information. This work describes using phase-angle information from tapping-mode AFM to follow the interactions of hydrogen with palladium, polycarbonate, and iron. Real-time AFM phase-angle information allowed for the observation of multiphase mass transfer to and from the surface of palladium at atmospheric pressure and room temperature without the need for complex sample preparation. The AFM observations are quantitatively benchmarked against and confirm mass transfer predictions based on bulk hydrogen diffusion data. Additionally, they support recent studies that demonstrate the existence of multiple hydrogen states during interactions with palladium surfaces.

Immobilization of enzymes on fumed silica nanoparticles for applications in nonaqueous media.

Enzymatic catalysis in nonaqueous media is considered as an attractive tool for the preparation of a variety of organic compounds of commercial interest. This approach is advantageous for numerous reasons including the enhanced stability of some substrates and products in solvents, sometimes improved selectivity of the enzyme, and reduction of unwanted water-dependent side reactions since little water is present. Due to the poor solubility of enzymes in these media, mass transfer limitations are sometimes present, leading to low apparent catalytic activity. Immobilization on solid supports has been successfully applied to overcome enzyme solubility issues by increasing the accessibility of substrates to the enzymes' active sites. We have developed a simple immobilization protocol that uses fumed silica as support. Fumed silica is an inexpensive nanostructured material with unique properties including large surface area and exceptional adsorptive affinity for organic macromolecules. Our protocol is performed in two main steps. First, the enzyme molecules are physically adsorbed on the surface of the non-porous fumed silica nanoparticles with the participation of silanol groups (Si-OH) and second, water is removed by lyophilization. The protocol has been successfully applied to both s. Carlsberg and Candida antarctica lipase B (CALB). The resulting fumed silica-based nanobiocatalysts of these two enzymes were tested for catalytic activity in hexane. The transesterification of N-acetyl-L: -phenylalanine ethyl ester was the model reaction for s. Carlsberg nanobiocatalysts. The simple esterification of geraniol and the enantioselective transesterification of (RS)-1-phenylethanol were the model reactions for CALB nanobiocatalysts. The observed catalytic activities were remarkably high and even exceeded those of commercially available preparations.

Enantioselective transesterification by Candida antarctica Lipase B immobilized on fumed silica.

Enzymatic catalysis to produce molecules such as perfumes, flavors, and fragrances has the advantage of allowing the products to be labeled "natural" for marketing in the U.S., in addition to the exquisite selectivity and stereoselectivity of enzymes that can be an advantage over chemical catalysis. Enzymatic catalysis in organic solvents is attractive if solubility issues of reactants or products, or thermodynamic issues (water as a product in esterification) complicate or prevent aqueous enzymatic catalysis. Immobilization of the enzyme on a solid support can address the generally poor solubility of enzymes in most solvents. We have recently reported on a novel immobilization method for Candida antarctica Lipase B on fumed silica to improve the enzymatic activity in hexane. This research is extended here to study the enantioselective transesterification of (RS)-1-phenylethanol with vinyl acetate. The maximum catalytic activity for this preparation exceeded the activity (on an equal enzyme amount basis) of the commercial Novozyme 435(®) significantly. The steady-state conversion for (R)-1-phenylethanol was about 75% as confirmed via forward and reverse reaction. The catalytic activity steeply increases with increasing nominal surface coverage of the support until a maximum is reached at a nominal surface coverage of 230%. We hypothesize that the physical state of the enzyme molecules at a low surface coverage is dominated in this case by detrimental strong enzyme-substrate interactions. Enzyme-enzyme interactions may stabilize the active form of the enzyme as surface coverage increases while diffusion limitations reduce the apparent catalytic performance again at multi-layer coverage. The temperature-, solvent-, and long-term stability for CALB/fumed silica preparations showed that these preparations can tolerate temperatures up to 70°C, continuous exposure to solvents, and long-term storage.

Conformational changes and catalytic competency of hydrolases adsorbing on fumed silica nanoparticles: II. Secondary structure.

Secondary conformational analysis via Circular Dichroism (CD) and Amide-I FTIR was applied to preparations of Candida antarctica Lipase B (CALB), subtilisin Carlsberg, and the Lipase from Thermomyces lanuginosus (TLL) on fumed silica to confirm that the "hardness" and packing density of the enzymes on the solid fumed silica nanoparticle surface can be used to rationalize the variable enzyme-dependent changes of catalytic competency with surface coverage. "Soft" enzymes should be immobilized at a surface coverage where enzyme-enzyme interactions predominate thereby preventing detrimental structural changes caused by enzyme-support interactions, while "hard" enzymes can be immobilized at low to intermediate surface coverage with good catalytic performance. Multi-layered coverage reduces the superficial average catalytic performance in all cases due to mass transfer limitations.

Conformational changes and catalytic competency of hydrolases adsorbing on fumed silica nanoparticles: I. tertiary structure.

We have recently introduced an immobilization protocol for preparations of enzymes on fumed silica for catalysis in organic solvents. The observation of a maximum in apparent catalytic activity at intermediate surface coverage for one enzyme while another enzyme showed continuously increasing apparent catalytic activity with decreasing surface coverage led to speculation on the impact of surface coverage on apparent catalytic activity through different relative surface-protein and protein-protein interactions, combined with different "hardness" or resistance towards unfolding by the enzymes. The kinetics of tertiary unfolding of Candida antarctica Lipase B (CALB), subtilisin Carlsberg, and the Lipase from Thermomyces lanuginosus (TLL) adsorbing on fumed silica nanoparticles were inferred here from tryptophan fluorescence for 2 to 1250%SC, 0.5-4.70 mg/mL enzyme concentration in aqueous buffer solution, and in the presence of the structural modifiers 2,2,2-trifluoroethanol (TFE) and dithiothreitol (DTT). The results shown here confirm the earlier speculation that "hard" enzymes can perform well at low and intermediate surface coverage of the solid fumed silica particles until multi-layer packing imposes mass-transfer limitations, while "soft" enzymes unfold at low surface coverage and therefore show a maximum in catalytic competency at intermediate surface coverage before declining apparent activity is caused by multi-layer packing.

Effect of water activity on the lipase catalyzed esterification of geraniol in ionic liquid [bmim]PF6.

Enzymatic reactions in non-aqueous media have been shown to be effective in carrying out chemical transformation where the reactants are insoluble in water or water is a byproduct limiting conversion. Ionic liquids, liquid organic salts with infinitesimal vapor pressure, are potentially useful alternatives to organic solvents. It is known that the thermodynamic water activity is an important variable affecting the activity of enzymes in non-aqueous solvents. This study investigated the influence of water activity on the esterification of geraniol with acetic acid in ionic liquid [bmim]PF6 catalyzed by immobilized Candida antarctica lipase B. The conversion of geraniol in [bmim]PF6 was significant although the reaction rate was slower than in organic solvents. The profile of initial reaction rate-water activity was determined experimentally, and differed from the data reported for other non-aqueous solvents. A maximum in the initial reaction rate was found at aw = 0.6. The pseudo reaction equilibrium constant, Kx, was measured experimentally for the reaction. The average value of Kx in [bmim]PF6 was 12, 20-fold lower than the value reported for the same system in hexane.