Electrochemical reduction of carbon dioxide and carbon monoxide for the production of green fuels and chemicals.

It has become apparent that closing the carbon cycle on this planet in order to mitigate disastrous consequences of runaway global warming has become one of the most pressing issues of our civilization. One of the ways we need to accomplish this goal is by finding news methods to generate fuels that will be carbon neutral. Renewable fuels and green chemicals will be a major component of closing the carbon cycle and restoring our planet’s ecosystem into a sense of balance. A method that can help achieve this goal is the reduction of CO2. If CO2 can be a desirable reactant on a large enough scale to produce fuels and chemicals, many industries will greatly benefit from the implementation of CO2 reduction technologies. This would in turn make removing CO2 from the atmosphere a worthy and realistic endeavor while reducing the concentration of CO2 in the atmosphere. Reducing CO2 concentrations in the atmosphere will help mitigate global warming. In the following dissertation, several methods are discussed that aid in the development of CO2 electroreduction. The goal of which is to improve the overall efficiency of current CO2 electroreduction technology. The first major effort assesses the contaminants emanating from the membrane component of the CO2 electroreduction vii device, alleviating the issue of spurious product detection. The second effort involves the tuning of product selectivity on oxide-derived copper catalyst by pulsing the bias. The third effort details the pursuit of a stand-alone “artificial leaf” technology for the reduction of carbon monoxide. The electrochemical investigations undertaken in this dissertation discuss in detail the metrics and principles used to accomplish these works

Increased Production and Extraction Efficiency of Triacylglycerides from Microorganisms and an Enhanced Understanding of the Pathways Involved in the Production of Triacylglycerides and Fatty Alcohols

The continued increase in the demand for fossil fuels combined with their ever dwindling supply has prompted the search for a suitable alternative fuel. The research contained within this dissertation seeks to increase the lipid content of cellular feedstocks, improve extraction efficiencies of lipids, and understand the pathways involved in the production of fatty alcohols and triacylglycerides from microbial feedstocks. As part of this research the diatom, Cheatoceros gracilis, was grown at small and large scale to determine optimal growing conditions. No apparent nutrient stress trigger was required to initiate the accumulation of the biodiesel precursor triacylglyceride, unlike other documented algal strains. A follow-up to this project demonstrated that the microalga C. gracilis may utilize light intensity as a trigger for lipid production. A major difficulty in the production of biofuels from microorganisms is the expensive process of dewatering, drying, and extracting the lipid compounds from the cells. As part of this research, a process has been developed that allows for lipid extraction to occur in the presence of water at a point as low as 2 percent solids or 98 percent water. This process utilizes a single organic solvent that mixes well with microbial lipids, but poorly with water allowing for efficient extraction of lipids and fast solvent to water separation. This process greatly decreases the cost of the microbial biofuels production associated with the removal of water from cell slurries. Triacylglycerides and fatty alcohols are oleochemicals that are commonly used in industrial, pharmaceutical, and consumable processes. A predicted fatty acyl CoA reductase enzyme was cloned into an E. coli vector, expressed, characterized and shown to be active as a dual reductive enzyme reducing a fatty acyl CoA to its respective fatty alcohol, constituting the first enzyme of this type discovered in a bacterium. The process of triacylglyceride production in microbes is fairly well understood; however, the process that regulates this production has not yet been fully explored. As part of this research, the model yeast organism, Yarrowea lipolytica, is utilized to identify essential genes for citrate transport that if removed could result in increasing triacylglyceride production in vivo

Development and Characterization of Poly(lactic acid)/Acetylated Starch Blends

In this study, the acetylation of starch was investigated as a means to improve its thermal processability and compatibility with poly(lactic acid) (PLA) and ultimately widen the range of potential applications for PLA/acetylated starch (AS) blends. The work was divided in two parts: (1) the characterization of AS according to degree of substitution (DS) and (2) the preparation and characterization of PLA/AS blends. The crystalline structure of AS, examined by X-ray diffraction, was destroyed upon acetylation. Differential scanning calorimetry (DSC) revealed that the removal of the crystalline structure during acetylation facilitated molecular movement and glass transition which should facilitate the thermal processability of AS. Decreased molecular weight of AS upon acetylation, which leads to the improvement of thermal processability, was confirmed by dilute solution viscometry. Thermogravimetric analysis (TGA) showed improved thermal stability upon acetylation which widens the processing temperature window of AS. Lower hydrophilicity of AS was revealed by contact angle analysis which is expected to generate more effective interactions with conventional polymers. Enhancement of the thermal expansion (measured by thermomechanical analysis (TMA)) and the lower density (measured by suspension) should improve the compatibility of AS with conventional polymers. The molecular mobility of AS with degree of substitution (DS) 1.5 and 3 was investigated by temperature-modulated DSC (TMDSC) and solid-state 13C NMR (SS-NMR). The relaxation time of hole formation, estimated from TMDSC data, was proposed as the representative of α-relaxation time near T_g. The T_g estimated from the VFT model was similar to the T_g measured by DSC and by heat-cool mode TMDSC supporting the analogy between the relaxation time of hole formation and the α-relaxation time. The cooperative rearranging region (CRR) size, investigated by heat-cool mode TMDSC, was smaller for DS3 compared to DS1.5 which was attributed to the disruption of hydrogen bonds. TMDSC experimental data at moderate temperature, predictions at ambient temperature and SS-NMR results at ambient temperature suggest different mobility of acetylated starch according to DS. The acetylation of starch generated PLA/AS blends with distinct properties. A biphasic morphology was predicted by the compressible regular solution (CRS) model and confirmed by scanning electron microscopy (SEM) observations for all PLA/AS blends, except the PLA/DS3 blend. The thickness of the interphase of the biphasic PLA/AS blends, estimated theoretically from the properties of the pure component and experimentally by small angle X-ray scattering (SAXS), increased with increasing DS of AS. The estimated interphase was generally related to the length scale of the dynamic heterogeneity of the PLA matrix. Slowdown of the PLA chain dynamics at the interphase region was estimated for PLA/DS0 and PLA/DS1.5 blends and related to the hydrogen bonding between the PLA chains and AS observed by FTIR. The properties of PLA/AS blends were affected by the DS of AS. The thermal stability of PLA/AS blends, obtained by TGA, was improved compared to neat PLA. DSC results indicated that AS had a plasticization and nucleation effect in the PLA/AS blends. The highest tensile strength, toughness, and impact strength was achieved when DS2.5 was added to PLA which may reflect the biphasic structure and the thick diffused interphase of this blend. The water diffusion coefficient and the water vapor permeability (WVP) of PLA/AS blends were influenced by the presence of AS. The water diffusion coefficient decreased while the WVP increased for PLA/AS blends by incorporation of AS. The rate of crystallization of neat PLA and PLA/AS blends with different DS were investigated. The PLA/DS0.5 and PLA/DS1.5 blends showed the highest and the lowest rate of crystallization in isothermal crystallization conditions, respectively. The highest rate of crystallization in isothermal crystallization conditions for the PLA/DS0.5 blend may reflect the highest nucleation activity of DS0.5 along with the faster dynamics of the PLA chains. In the context of the PLA/DS1.5 blend, the highest initial degree of crystallinity may seem contrary with the lowest crystallization rate of this blend in isothermal crystallization conditions. The non-isothermal crystallization investigation, however, revealed similar rates of crystallization for the PLA/DS1.5 blend compared to the other PLA/AS blends at the lowest cooling rate (2°C/min) while having the fastest rate of crystallization at the highest cooling rate (5°C/min). The positive activation energy for the crystallization of the PLA/DS1.5 blend may reflect an endothermic process in the crystallization of this blend as a result of interfacial interactions. The largest amorphous phase thickness observed by SAXS for the PLA/DS1.5 blend could explain the significance of the interfacial interactions in this blend. Finally, the results of this study showed that the acetylation of starch is a promising avenue for PLA/starch blends. Increasing the processability of starch and its compatibility with PLA through acetylation, provides the opportunity of making a wide range of materials with different applications. Various mechanical, thermal, and water transport properties, transparency and crystallization behavior can be achieved by incorporating AS with different DS into PLA. For final applications, the effect of AS concentration on the properties of the PLA/AS blend should be investigated. The concentration of the AS dispersed phase in PLA/AS blends should lead to differences in morphology, interfacial characteristics and ultimately properties of the blend. Transparency, visual appearance and degradation rate of the blends are also very important factors in final applications that need to be studied in the future

Structure and function of glutamate transporters

The work presented in the following chapters primarily involves determining specific structural and functional features involved with glutamate transport by the excitatory amino acid transporters (EAAT1-5). This research derives from a study of X-ray crystal structures of an homologous archaeal transporter. Broadly, using computational methods to probe EAAT homology models born from the archaeal structures, we introduce/describe novel Na+ and K+ coordination sites and present a model for sequential cation and substrate binding and unbinding during the transport cycle. A secondary focus of this dissertation was computational modeling of two other proteins: the HIV gp120 envelope protein (gp120) and NAD(P)H:Quinone Oxidoreductase 1 (NQO1). The study of gp120 involved a statistical analysis of substitutions that occur during early infection of clade A HIV, showing that during early infection substitutions are non-random and co-locate in regions associated with receptor binding. The computational component of the study involving NQO1 used computational molecular docking of two lavendamycin analogues into the NQO1 active site. Here we showed that relative scores of docked poses of the analogues were consistent with in vitro evaluations. All the studies presented in this dissertation were collaborative efforts that linked in silico work with in vitro analysis

Hydrogenative depolymerization of nylons

This research was supported by the European Research Council (ERC AdG 692775). D. M. holds the Israel Matz Professorial Chair of Organic Chemistry. A. K. is thankful to the Planning and Budgeting Committee of Israel and Feinberg Graduate School for a (senior) postdoctoral fellowship. Y.-Q. Z. acknowledges the Sustainability and Energy ResearchInitiative (SAERI) foundation for a research fellowship. Computations were performed using HPC resources from GENCI-CINES (Grant 2019 AP010811227).The widespread crisis of plastic pollution demands discovery of new and sustainable approaches to degrade robust plastics such as nylons. Using a green and sustainable approach based on hydrogenation, in the presence of a ruthenium pincer catalyst at 150 oC and 70 bar H2, we report here the first example of hydrogenative depolymerization of conventional, widely used nylons, and polyamides in general. Un-der the same catalytic conditions, we also demonstrate the hydrogenation of a polyurethane to produce diol, diamine and methanol. Additionally, we demonstrate an example where monomers (and oligomers) obtained from the hydrogenation process can be dehydrogenated back to a poly(oligo)amide of approximately similar molecular weight, thus completing a closed loop cycle for recycling of poly-amides. Based on the experimental and DFT studies, we propose a catalytic cycle for the process that is facilitated by metal-ligand cooperativity. Overall, this unprecedented transformation, albeit at the proof of concept level, offers a new approach towards a cleaner route to recycling nylons.Publisher PDFPeer reviewe

Bioactive Recombinant Human Oncostatin M for NMR-Based Screening in Drug Discovery

Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis. Recently, elevated OSM levels have been found in the serum of COVID-19 patients in intensive care units. Multiple anti-OSM therapeutics have been investigated, but to date no OSM small molecule inhibitors are clinically available. To pursue a high-throughput screening and structure-based drug discovery strategy to design a small molecule inhibitor of OSM, milligram quantities of highly pure, bioactive OSM are required. Here, we developed a reliable protocol to produce highly pure unlabeled and isotope enriched OSM from E. coli for biochemical and NMR studies. High yields (ca. 10 mg/L culture) were obtained in rich and minimal defined media cultures. Purified OSM was characterized by mass spectrometry and circular dichroism. The bioactivity was confirmed by induction of OSM/OSM receptor signaling through STAT3 phosphorylation in human breast cancer cells. Optimized buffer conditions yielded 1H, 15N HSQC NMR spectra with intense, well-dispersed peaks. Titration of 15N OSM with a small molecule inhibitor showed chemical shift perturbations for several key residues with a binding affinity of 12.2 ± 3.9 μM. These results demonstrate the value of bioactive recombinant human OSM for NMR-based small molecule screening

Effects of açaí berry (Euterpe oleracea) extracts on human antioxidant systems and drug metabolism

The source and sink of reactive oxygen species are diverse and so are the control mechanisms to counteract them. Sources may be exogenous or endogenous from normal metabolic pathways. Similarly, the sink could be a simple radical scavenging event by small molecule antioxidants and antioxidant enzymes or complex events involving cellular signaling processes. The effects of reactive oxygen species may precipitate cellular dysfunction from its toxicity, protect from invading microorganisms or perform essential functions through regulation of cell signaling pathways. Excessive Reactive oxygen species leads to oxidative stress which mediates cellular damage and is implicated in several pathological conditions. The current research addresses biological systems that may effect redox balance in humans. One goal of the current research was to establish structure activity relationship for substrate binding to several human drug metabolizing enzymes implicated in reactive oxygen species generation, namely CYP2A6 and CYP2E1. The substrate dynamics of these two enzymes were studied by probing the active site with a series of small chain saturated and 2,3-unsaturated aldehydes using human liver microsomes. The study demonstrated that the aldehydes inhibited both the enzymes in competitive manner with unsaturated aldehydes being more potent than their saturated counterparts. The potential for p-stacking interactions between the phenylalanine rich active site of these enzymes and the double bond at 2-position in unsaturated aldehydes conferred high affinity for these aldehydes. It also confirmed an earlier findings that the active site of CYP2A6 is rigid where as CYP2E1 is flexible due to the presence of extended p-system allowing the expansion of its active site. Another goal was to examine the possible interactions between human cytochrome P450 enzymes of pharmacological and toxicological importance with a natural product called açaí. Açaí (Euterpe oleracea) is a Brazilian palm tree that has emerged from traditional medicinal plant to a recent super-fruit status. The assumption that it is safe to consume açaí currently lacks evidence from its interactions with drug metabolizing cytochrome P450 enzymes. The interaction between the crude extracts of açaí and major cytochrome P450 enzymes involved in drug metabolism and toxicology demonstrated the potential for chloroform extract to inhibit the isoforms CYP1A1, CYP2B6 and CYP2C8. The Michaelis-Menten Kinetics studies indicated mixed mode of inhibition of these enzymes by crude chloroform extract of açaí with low KI and KI’ values for CYP2C8 followed by CYP2B6 and CYP1A1. In addition, the study was extended to identification of inhibitors for toxicologically important CYP2A6 and CYP2E1 using a collaborative bioassay-guided fractionation approach. Although the crude chloroform extract of açaí showed considerable inhibition of these enzymes, specific inhibitors were not identified. Finally, the regulation of a signal transduction pathway namely Nrf2/ARE signaling pathway by açaí constituents was also studied as a potential strategy to prevent oxidative damage. The nuclear factor erythoid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway is a cellular defense to counteract oxidative stress. Activation of this pathway increases the expression of a battery of antioxidant genes. This was achieved by monitoring the activation of a cis¬-acting DNA sequence referred to as Antioxidant Response Element (ARE) contained in a luciferase-containing promoter vector in cultured HepG2 cells. A high-throughput analysis of fractions generated using bioassay-guided fractionation of açaí has resulted in the identification of a class of compounds known as Pheophorbides as the inducers of ARE-luciferase. Dose response analysis using pure compounds demonstrated significant induction of ARE-luciferase at concentrations as low as 8.2 µM and 16.9µM for Pheophorbide a methyl ester and Pheophorbide a, respectively

How Water's Properties Are Encoded in Its Molecular Structure and Energies.

How are water's material properties encoded within the structure of the water molecule? This is pertinent to understanding Earth's living systems, its materials, its geochemistry and geophysics, and a broad spectrum of its industrial chemistry. Water has distinctive liquid and solid properties: It is highly cohesive. It has volumetric anomalies-water's solid (ice) floats on its liquid; pressure can melt the solid rather than freezing the liquid; heating can shrink the liquid. It has more solid phases than other materials. Its supercooled liquid has divergent thermodynamic response functions. Its glassy state is neither fragile nor strong. Its component ions-hydroxide and protons-diffuse much faster than other ions. Aqueous solvation of ions or oils entails large entropies and heat capacities. We review how these properties are encoded within water's molecular structure and energies, as understood from theories, simulations, and experiments. Like simpler liquids, water molecules are nearly spherical and interact with each other through van der Waals forces. Unlike simpler liquids, water's orientation-dependent hydrogen bonding leads to open tetrahedral cage-like structuring that contributes to its remarkable volumetric and thermal properties

Structural Stabilization of α-Helical Antifreeze Protein Variants Using the Trp-cage Protein

Antifreeze proteins are prevalent in many species that experience freezing or close to freezing temperatures. The proteins lower the freezing point of water, enabling the species to survive subzero temperatures. One of these species is fish, in which antifreeze proteins originally were discovered. While antifreeze proteins can be found in a wide variety of secondary and tertiary structures, the α helical antifreeze protein from winter flounder (WflAFP) is probably the most extensively studied. The antifreeze activity of all antifreeze proteins has been correlated to size and flatness of the ice-binding surface which for WflAFP has been demonstrated to be on one side of the alpha-helix, comprising primarily regularly spaced alanine and threonine side chains. The analysis of the size necessary for activity, however, was complicated in the case of WflAFP as a shortening of the sequence also lead to a reduction in α helicity. To overcome this difficulty, in this work a C-terminal stabilizing and alpha-helicity inducing capping unit, the Trp-cage, was employed. In a first step, the effect of the C-terminal modification on the wild-type antifreeze protein had to be studied to eliminate any interference of the modification on ice activity. In a second step, after eliminating any negative influence of the capping unit, the dependence of ice activity on the size of the ice-binding surface could be analyzed. The combination of both Trp-cage and antifreeze protein segment was pursued using two different approaches: The fusion approach was based on the synthesis of both the Trp-cage capping unit and an antifreeze protein segment separately and fusing them together in a second step. The chimera approach is based on the fact, that in the primary sequence of both Trp-cage and antifreeze protein some amino acids are interchangeable leading to an overlapping sequence and overall a shorter protein. Four alpha-helical antifreeze protein variants of different lengths were stabilized using the Trp-cage. Of the four ice-binding Trp-cage chimera (IBTC), three were also obtained as GFP labeled variants. All IBTC were characterized using NMR , CD , and UV/vis spectroscopy, mass spectrometry and their antifreeze activity or ice growth retardation as well as ice shaping abilities were determined. All ice-binding Trp-cage chimera designed based on the chimera approach were well folded. The IBTC designed based on the fusion approach, on the other hand, had a less pronounced fold according to 1H NMR data. All folded IBTC also satisfied the criteria for α helicity in a chemical shift deviation plot and the CD data also indicate α helicity for all IBTC. The analysis of ice activity gave surprising results. The IBTC based on the wild-type antifreeze protein had comparable antifreeze activity to WflAFP. However, no antifreeze activity could be observed for the other IBTC. As a result, even though the C-terminal capping unit has no influence on the antifreeze activity of the IBTC, the induction and stabilization of α helicity is not the only criteria necessary for activity. Rather, the size of the ice-binding surface has to match the one present in nature to generate antifreeze activity