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Despite its massive potential, Raman imaging represents just a modest fraction of all research and clinical microscopy to date. This is due to the ultralow Raman scattering cross-sections of most biomolecules that impose low-light or photon-sparse conditions. Bioimaging under such conditions is suboptimal, as it either results in ultralow frame rates or requires increased levels of irradiance. Here, we overcome this tradeoff by introducing Raman imaging that operates at both video rates and 1,000-fold lower irradiance than state-of-the-art methods. To accomplish this, we deployed a judicially designed Airy light-sheet microscope to efficiently image large specimen regions. Further, we implemented subphoton per pixel image acquisition and reconstruction to confront issues arising from photon sparsity at just millisecond integrations. We demonstrate the versatility of our approach by imaging a variety of samples, including the three-dimensional (3D) metabolic activity of single microbial cells and the underlying cell-to-cell variability. To image such small-scale targets, we again harnessed photon sparsity to increase magnification without a field-of-view penalty, thus, overcoming another key limitation in modern light-sheet microscopy.
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Iluminação , Microscopia , Microscopia/métodos , Fótons , Imageamento Tridimensional/métodosRESUMO
More than 23 million tonnes of lignin are produced annually in the US from wood pulping and 98% of this lignin is burnt. Therefore, creating products from lignin, such as plastics, offers an approach for obtaining sustainable materials in a circular economy. Lignin-based copolymers were synthesized using a single pot, solvent free, melt condensation reaction. The synthesis occurred in two stages. In the first stage, a biobased prepolymer consisting of butanediol (BD, 0.8-1 molar content) and a diacid (succinic (SA), adipic (AA) and suberic acids (SuA), with varying amounts of diaminobutane (DAB, 0-0.2 molar content) was heated under vacuum and monitored by Fourier transform infra-red (FTIR) spectroscopy and electrospray ionization-mass spectrometry (ESI-MS). In the second stage, prepolymer was mixed with a softwood kraft lignin (0-50 wt.%) and further reacted under vacuum at elevated temperature. Progression of the polymerization reaction was monitored using FTIR spectroscopy. The lignin-copolyester/amide properties were characterized using tensile testing, X-ray diffraction (XRD), dynamic mechanical analysis (DMA), differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) techniques. Lignin co-polymer tensile (strength 0.1-2.1 MPa and modulus 2 to 338 MPa) properties were found to be influenced by the diacid chain length, lignin, and DAB contents. The lignin-copolymers were shown to be semi-crystalline polymer and have thermoplastic behavior. The SA based copolyesters/amides were relatively stiff and brittle materials while the AA based copolyesters/amides were flexible and the SuA based copolyesters/amides fell in-between. Additionally, > 30 wt.% lignin the lignin- copolyesters/amides did not exhibit melt behavior. Lignin-co-polyester/amides can be generated using green synthesis methods from biobased building blocks. The lignin- copolyesters/amides properties could be tuned based on the lignin content, DAB content and diacid chain length. This approach shows that undervalued lignin can be used in as a macromonomer in producing thermoplastic materials.
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Amidas/química , Lignina/química , Plásticos/química , Poliésteres/química , Fenômenos Químicos , Estrutura Molecular , Polimerização , Espectrometria de Massas por Ionização por Electrospray , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Resistência à Tração , Termogravimetria , Difração de Raios XRESUMO
The process of wood formation is of great interest to control and manipulate wood quality for economically important gymnosperms. A Douglas-fir tissue culture system was developed that could be induced to differentiate into tracheary elements (fibers) making it possible to monitor xylogenesis in vitro by a proteomics approach. Two proteomes were analyzed and compared, one from an early and one from a late stage of the fiber differentiation process. After 18 weeks in a differentiation-inducing medium, 80% of the callus cells were elongated while 20% showed advanced spiral thickening indicating full wood fiber differentiation. Based on 2D electrophoresis, MS, and data analyses (data are available via ProteomeXchange with identifier PXD001484.), it was shown that in nondifferentiated callus (representing an early stage of development), proteins related to protein metabolism, cellular energy, and primary cell wall metabolism were abundant. By comparison, in cells actively differentiating wood fibers (representing a late stage of development), proteins involved in cell wall polysaccharide biosynthesis predominated together with housekeeping and stress-associated proteins.
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Proteínas de Plantas/análise , Proteoma/análise , Pseudotsuga/crescimento & desenvolvimento , Madeira/crescimento & desenvolvimento , Eletroforese em Gel Bidimensional , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteoma/química , Proteoma/metabolismo , Pseudotsuga/química , Pseudotsuga/metabolismo , Técnicas de Cultura de Tecidos , Madeira/química , Madeira/metabolismoRESUMO
Polyhydroxybutyrate (PHB) was grafted onto cellulose fiber by dicumyl peroxide (DCP) radical initiation via in situ reactive extrusion. The yield of the grafted (cellulose-g-PHB) copolymer was recorded and grafting efficiency was found to be dependent on the reaction time and DCP concentration. The grafting mechanism was investigated by electron spin resonance (ESR) analysis and showed the presence of radicals produced by DCP radical initiation. The grafted copolymer structure was determined by nuclear magnetic resonance (NMR) spectroscopy. Scanning electronic microscopy (SEM) showed that the cellulose-g-PHB copolymer formed a continuous phase between the surfaces of cellulose and PHB as compared to cellulose-PHB blends. The relative crystallinity of cellulose and PHB were quantified from Fourier transform infrared (FTIR) spectra and X-ray diffraction (XRD) results, while the absolute degree of crystallinity was evaluated by differential scanning calorimetry (DSC). The reduction of crystallinity indicated the grafting reaction occurred not just in the amorphous region but also slightly in crystalline regions of both cellulose and PHB. The smaller crystal sizes suggested the brittleness of PHB was decreased. Thermogravimetric analysis (TGA) showed that the grafted copolymer was stabilized relative to PHB. By varying the reaction parameters the compositions (%PHB and %cellulose) of resultant cellulose-g-PHB copolymer are expected to be manipulated to obtain tunable properties.
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Compostos de Benzil/química , Celulose/química , Hidroxibutiratos/química , Acetilação , Configuração de Carboidratos , Cristalografia por Raios X , Radicais Livres/químicaRESUMO
Lignin-based triple shape memory polymers comprised of both permanent covalent cross-links and physical cross-links have been synthesized. A mixing phase with poly(ester-amine) and poly(ester-amide) network having two distinct glass transitions was hot mixed with more structurally homogenized methanol soluble lignin fraction by one-pot, two-step method. Triple shape properties arise from the combined effect of the glass transition of polyester copolymers and lignin and the dissociation of self-complementary hydrogen bonding and cross-link density. The percentage of recovery in each stage was investigated and it was proved that the first recovery is related with lignin-poly(ester-amine) rich network and the second recovery stage is related with lignin-poly(ester-amide) rich network. The thermal and mechanical properties of the lignin-copolymer networks were also investigated using differential scanning calorimetry and dynamic mechanical analysis.
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Lignina/química , Nylons , Poliaminas , Poliésteres , Nylons/síntese química , Nylons/química , Poliaminas/síntese química , Poliaminas/química , Poliésteres/síntese química , Poliésteres/químicaRESUMO
Stalk lodging (structural failure crops prior to harvest) significantly reduces annual yields of vital grain crops. The lack of standardized, high throughput phenotyping methods capable of quantifying biomechanical plant traits prevents comprehensive understanding of the genetic architecture of stalk lodging resistance. A phenotyping pipeline developed to enable higher throughput biomechanical measurements of plant traits related to stalk lodging is presented. The methods were developed using principles from the fields of engineering mechanics and metrology and they enable retention of plant-specific data instead of averaging data across plots as is typical in most phenotyping studies. This pipeline was specifically designed to be implemented in large experimental studies and has been used to phenotype over 40,000 maize stalks. The pipeline includes both lab- and field-based phenotyping methodologies and enables the collection of metadata. Best practices learned by implementing this pipeline over the past three years are presented. The specific instruments (including model numbers and manufacturers) that work well for these methods are presented, however comparable instruments may be used in conjunction with these methods as seen fit.â¢Efficient methods to measure biomechanical traits and record metadata related to stalk lodging.â¢Can be used in studies with large sample sizes (i.e., > 1,000).
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In this work we report on the preparation, characterization, and properties of a thermally treated lignin-derived, phenolic-rich fraction (PRF) of wood pyrolysis bio-oil obtained by ethyl acetate extraction. The PRF was characterized for viscoelastic and rheological behavior using dynamic mechanical analysis (DMA) and cone and plate rheology. A unique thermoplastic behavior was evidenced. Heat-treated PRFs acquire high modulus but show low temperatures of thermal flow which can be systematically manipulated through the thermal pretreatment. Loss of volatiles, changes in molecular weight, and glass transition temperature (Tg) were investigated using thermogravimetric analysis (TGA), mass spectrometry (MS), and differential scanning calorimetry (DSC), respectively. Underlying mechanisms for the thermal and rheological behavior are discussed with regard to interactions between pyrolytic lignin nanoparticles present in the system and the role of volatile materials on determining the properties of the material resembling in several aspects to colloidal suspension systems. Low thermal flow temperatures and reversible thermal effects can be attributed to association of pyrolytic lignin particles due to intermolecular interactions that are easily ruptured at higher temperatures. The thermoplastic behavior of PRF and its low Tg is of particular interest, as it gives opportunities for application of this fraction in several melt processing and adhesive technologies.
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Óleos/química , Fenol/análise , Madeira/química , Adesivos/química , Materiais Biocompatíveis , Varredura Diferencial de Calorimetria , Coloides/química , Temperatura Alta , Lignina/química , Espectrometria de Massas , Peso Molecular , Nanopartículas , Fenol/química , Reologia , Termogravimetria , Temperatura de TransiçãoRESUMO
The aim of this study was to evaluate the use of waste natural fibers from milled hop bines and hemp stalks, without chemical treatment, and compare them to a commercial wood fiber for use in wood-plastic composite (WPC) materials. The fibers were characterized (density, fiber size and chemical composition). WPCs were produced by the extrusion of a blend of fibers (50%), high-density polyethylene (HDPE) and coupling agent (2%). The WPCs were characterized for their mechanical, rheological, thermal, viscoelastic and water resistance properties. Pine fiber was about half the size of hemp and hop fibers and thus had a higher surface area. The pine WPC melts had a higher viscosity than the other two WPCs. Additionally, the tensile and flexural strengths of the pine WPC were higher than those of hop and hemp WPCs. The pine WPC was also shown to have the least water absorption followed by hop and hemp WPCs. This study highlights that different lignocellulosic fibers influence their WPC properties. The properties of the hop- and hemp-based WPCs were comparable to commercial WPCs and can be improved by further milling/screening the fibers to a smaller particle size (volumetric mean of ~88 µm) to increase their surface area, fiber-matrix interactions and improve stress-transfer.
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Biomass and its interactions for heat generation have received little attention. In this study, the woody biomass materials were Prosopis africana (PA), Harungana madascariences (HM), Vitrllaria paradoxa (VP), and Afzelia africana (AA). The composition (extractives, carbohydrate, and lignin) of the biomass was determined. The biomass was converted to charcoal in a traditional kiln. A thermo-kinetic examination of the charcoal samples was carried out. The kinetic parameters and potential reaction mechanisms involved in the decomposition process were both obtained using the integral (Flynn-Wall Ozawa) isoconversional methods in conjunction with the Coats-Redfern approach. The activation energy profiles for the charcoal samples in oxidizing atmospheres were 548 kJ/mol for AA, 274 kJ/mol for VP, 548 kJ/mol for PA, and 274 kJ/mol for HM. All charcoal samples underwent comprehensive, multi-step, complex reaction pathways for thermal degradation. The charcoal samples exhibit not only great potential for biochemical extraction but also for bioenergy applications. The significant amount of combustion characteristics in the raw biomass and charcoal samples indicates that each type of wood charcoal produced has more fixed carbon, less ash, and less volatile matter, all of which are desirable for the thermo-chemical conversion of biomass for the production of heat.
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Carvão Vegetal , Temperatura Alta , Carvão Vegetal/química , Biomassa , Madeira , Cinética , TermogravimetriaRESUMO
Most research in plants and other organisms has, for the sake of convenience, focused on the use of model species to identify mechanisms that are conserved throughout the whole kingdom. Nevertheless, unique features and processes such as those related to plant cell wall and fiber formation, and to wood quality, sometimes need to be studied directly in the non-model organism of interest. Such organisms, like the economically and ecologically important gymnosperm Douglas-fir (Pseudotsuga menziesii), which is one of the crucial softwood timber species in Northern America, are often difficult to investigate. High phenolic, resin, and tannin contents in the woody tissues, as well as an incompletely sequenced genome, have contributed greatly to the species' recalcitrance for molecular biology investigations. In this study, we present a complete procedure detailing protein sample preparation, separation, and proteomic analysis based on cross-species identification of Douglas-fir. Proteins from the cambial zone, mature needles, and in vitro callus were extracted, purified, and separated via 1D and 2D SDS-PAGE. One-dimensional electrophoresis coupled with ESI-MS/MS was used for cross-species protein identification in order to evaluate the potential of this approach and reveal major differences in protein profiles among tested tissues. Identified proteins were functionally and developmentally compared. The likely contribution of these proteins to the properties of the cell wall and wood is indicated and discussed.
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Proteínas de Plantas/análise , Proteoma/análise , Pseudotsuga/química , Eletroforese em Gel Bidimensional , Proteínas de Plantas/química , Proteoma/química , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Madeira/químicaRESUMO
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with various 3-hydroxyvalerate (3HV) contents biosynthesized by mixed microbial consortia (MMC) fed fermented dairy manure at the large-scale level was assessed over a 3-month period. The thermal, mechanical, and rheological behavior and the chemical structure of the extracted PHBV biopolymers were studied. The recovery of crude PHBV extracted in a large Soxhlet extractor with CHCl3 for 24 h ranged between 20.6% to 31.8% and purified to yield between 8.9% to 26.9% all based on original biomass. 13C-NMR spectroscopy revealed that the extracted PHBVs have a random distribution of 3HV and 3-hydroxybutyrate (3HB) units and with 3HV content between 16% and 24%. The glass transition temperature (Tg) of the extracted PHBVs varied between -0.7 and -7.4 °C. Some of the extracted PHBVs showed two melting temperatures (Tm) which the lower Tm1 ranged between 126.1 °C and 159.7 °C and the higher Tm2 varied between 152.1 °C and 170.1 °C. The weight average molar mass of extracted PHBVs was wide ranging from 6.49 × 105 g·mol-1 to 28.0 × 105 g·mol-1. The flexural and tensile properties were also determined. The extracted polymers showed a reverse relationship between the 3HV content and Young's modulus, tensile strength, flexural modulus, and flexural strength properties.
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Single-cells grow by increasing their biomass and size. Here, we report that while mass and size accumulation rates of single Escherichia coli cells are exponential, their density and, thus, the levels of macromolecular crowding fluctuate during growth. As such, the average rates of mass and size accumulation of a single cell are generally not the same, but rather cells differentiate into increasing one rate with respect to the other. This differentiation yields a density homeostasis mechanism that we support mathematically. Further, we observe that density fluctuations can affect the reproduction rates of single cells, suggesting a link between the levels of macromolecular crowding with metabolism and overall population fitness. We detail our experimental approach and the "invisible" microfluidic arrays that enabled increased precision and throughput. Infections and natural communities start from a few cells, thus, emphasizing the significance of density-fluctuations when taking non-genetic variability into consideration.
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Escherichia coli , Reprodução , Escherichia coli/metabolismo , Homeostase , Substâncias Macromoleculares/metabolismoRESUMO
The cell wall compositional (lignin and polysaccharides) variation of two sweet sorghum varieties, Della (D) and its variant REDforGREEN (RG), was evaluated at internodes (IN) and nodes (N) using high-performance liquid chromatography (HPLC), pyrolysis-gas chromatography-mass spectrometry (Py-GCMS), X-ray diffraction (XRD), and two-dimensional (2D) 1H-13C nuclear magnetic resonance (NMR). The stalks were grown in 2018 (D1 and RG1) and 2019 (D2 and RG2) seasons. In RG1, Klason lignin reductions by 16-44 and 2-26% were detected in IN and N, respectively. The analyses also revealed that lignin from the sorghum stalks was enriched in guaiacyl units and the syringyl/guaiacyl ratio was increased in RG1 and RG2, respectively, by 96% and more than 2-fold at IN and 61 and 23% at N. The glucan content was reduced by 23-27% for RG1 and by 17-22% for RG2 at internodes. Structural variations due to changes in both cellulose- and hemicellulose-based sugars were detected. The nonacylated and γ-acylated ß-O-4 linkages were the main interunit linkages detected in lignin. These results indicate compositional variation of stalks due to the RG variation, and the growing season could influence their mechanical and lodging behavior.
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Sorghum , Parede Celular , Cromatografia Gasosa-Espectrometria de Massas , Lignina , Espectroscopia de Ressonância MagnéticaRESUMO
Peas are an underutilized crop that do not require allergen labeling and are rarely genetically modified. Peas contain less protein than soy and vary in protein composition. Because peas contain more starch than soy and less lipids, an alternative procedure for pea tofu production needs to be developed to prevent excessive starch gelatinization while promoting curd development. To accomplish this, a response surface model design was utilized to determine optimal oil addition, cook time, and salt concentration. Treatment ranges were from 0.0% to 4.2% for oil addition, 60-134 min for cook time, and 5.0%-9.2% for MgCl2 addition. Treatments had varying effects on tofu texture. Cook time was directly proportional to the hardness and could be used to match the soft, firm, and extra firm texture targets of conventional soy tofu. Protein secondary structure was not related to gel strength, indicating a system with synergies between multiple components other than protein. This research will help satisfy the growing demand for alternatives to soy-based foods.
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A non-isothermal decomposition of Moringa oleifera husk and Delonix regia seed pod was carried out in an N2 pyrolytic condition with the primary objective of undertaking the kinetics modeling, thermodynamics and thermal performance analyses of the identified samples. Three different isoconversional models, namely, differential Friedman, Flynn-Wall-Ozawa, and Starink techniques were utilized for the deduction of the kinetics data. The thermodynamic parameters were deduced from the kinetic data based on a first-order chemical reaction model. In the kinetics study, a strong correlation (R2 > 0.9) was observed throughout the conversion range for all the kinetic models. The activation energy profiles showed two distinctive regions. In the first region, the average activation energy values were relatively higher-a typical example is in the Flynn-Wall-Ozawa technique-MH (199 kJ/mol) and RP (194 kJ/mol), while in the second region, MH (292 kJ/mol) and RP (234 kJ/mol). It was also demonstrated that the thermal process for the samples experienced endothermic reactions thought the conversion range. In summary, both the kinetic and thermodynamic parameters vary significantly with conversion-underscoring the complexity associated with the thermal conversion of lignocellulosic biomass samples.
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Understanding the properties and flow characteristics of recycled polyolefins in rice hull composite blends is of importance to facilitate process optimization whilst promoting sustainability. The influence of milled rice hull particle size (<0.5 mm and <1 mm) on properties of recycled polyolefins composites was studied with major focus on recycled high-density polyethylene (rHDPE) and polypropylene (rPP) together with added maleated polymer coupling agents. Composites were compounded/extruded using a twin-screw extruder and the thermal, mechanical, and physical properties were analyzed as well as their melt flow, dynamic. and capillary rheology tests. The incorporation of the <0.5 mm rice-hulls particles enhanced the composite properties of viscosity, flexural strength, moduli, water absorption, and thermal stability for both polyolefins with rHDPE composites showing more reliable properties as compared to rPP.
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With the continued growing U.S. population, solid waste generation will increase, which will lead to undesired and significant growth in landfilling. Thermal treatment can turn these high calorific value wastes into fuels that can be used in small-to-large power plants. This article focuses on using blends with 40% plastic and 60% fiber wastes and converting them into densified solid fuel by torrefaction and extrusion. The material was torrefied at 300 °C to obtain torrefied samples with different mass losses, ranging from 0% to a maximum of 51%. The torrefaction results showed a clear synergy between plastics and fibers. The torrefied material was then extruded into 9 mm diameter rods and the products were characterized by molecular functional group analysis, thermomechanical analysis, dynamic mechanical analysis, dynamic rheological measurement, density measurement, flexural testing, water absorption test, size distribution measurement, heat content test, and combustion test. The fiber content in the material decreased as mass loss increased, and the process reduced significantly the variability of the material. The heat content increased as the mass loss increased. The plastic in the feedstock acted as a process enabler as it imparted properties like bindability, water resistance, high heat content, and increased degradation reaction rate.
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Temperatura Alta , Resíduos Sólidos , Biomassa , Plásticos , Temperatura , ÁguaRESUMO
Tofu, made by coagulating soy milk, is a nutritious food originating in China and is widely consumed globally. Due to allergenicity and consumer perceptions of genetically modified organisms, consumer demand for soy alternatives is increasing. In this study, tofu was made from yellow split peas (Pisum sativum). Effects of pasteurization, fat addition, and curd disruption followed by repressing were studied. Here, disruption was not a chemical disruption, rather a physical disruption of protein curds. Pasteurization alone led to increased uptake of water and nonviable tofus. Disrupted samples became firmer with pasteurization. Texture profile analysis indicated that disruption followed by pasteurization improved hardness from approximately 175 g force from the control, to approximately 325 g force for disrupted + pasteurizated samples without fat addition. A similar trend was observed for samples with fat added, where hardness increased from approximately 50 g force to approximately 75 g force. Fourier-transform infrared spectroscopy of the amide I region showed that an increase of ß-sheet structures led to increased hardness. The shifts in ß-sheet structures followed the same trends as surface hydrophobicity. Surface hydrophobicity decreased with pasteurization and increased with disruption. Molecular weight analysis showed that shear (from disruption) and heat separately degraded the proteins into smaller polypeptides exposing hydrophobic interiors. Changes to biochemical parameters, such as protein secondary structure and exposure of protein hydrophobic regions, allowed for tofu to be made from yellow field peas. PRACTICAL APPLICATION: This study provides critical information and a means to produce pea-based soy-free tofu.
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Manipulação de Alimentos/métodos , Pisum sativum/química , Preparações de Plantas/química , Culinária , Dureza , Temperatura Alta , Interações Hidrofóbicas e HidrofílicasRESUMO
The effects of Al2O3 coating on the performance of silica nanospring (NS) supported Co catalysts for Fischer-Tropsch synthesis (FTS) were evaluated in a quartz fixed-bed microreactor. The Co/NS-Al2O3 catalysts were synthesized by coating the Co/NS and NS with Al2O3 by an alkoxide-based sol-gel method (NS-Al-A and NS-Al-B, respectively) and then by decorating them with Co. Co deposition was via an impregnation method. Catalysts were characterized before the FTS reaction by the Brunauer-Emmett-Teller (BET) method, X-ray diffraction, transmission electron microscopy, temperature programmed reduction, X-ray photoelectron spectroscopy, differential thermal analysis and thermogravimetric analysis in order to find correlations between physico-chemical properties of catalysts and catalytic performance. The products of the FTS were trapped and analyzed by GC-TCD and GC-MS to determine the CO conversion and reaction selectivity. The Al2O3 coated NS catalyst had a significant affect in FTS activity and selectivity in both Co/NS-Al2O3 catalysts. A high CO conversion (82.4%) and Σ > C6 (86.3%) yield were obtained on the Co/NS-Al-B catalyst, whereas the CO conversion was 62.8% and Σ > C6 was 58.5% on the Co/NS-Al-A catalyst under the same FTS experimental condition. The Co/NS-Al-A catalyst yielded the aromatic selectivity of 10.2% and oxygenated compounds.
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Previous research has demonstrated that production of natural fiber reinforced thermoplastic composites (NFRTCs) utilizing bacterially-derived pure polyhydroxybutyrate (PHB) does not yield a product that is cost competitive with synthetic plastic-based NFRTCs. Moreover, the commercial production of pure PHB is not without environmental impacts. To address these issues, we integrated unpurified PHB in NFRTC construction, thereby eliminating a significant energy and cost sink (ca. 30-40%) while concurrently yielding a fully biologically based commodity. PHB-rich biomass synthesized with the microorganism Azotobacter vinelandii UWD was utilized to manufacture NFRTCs with wood flour. Resulting composites exhibited statistically similar bending strength properties despite relatively different PHB contents. Moreover, the presence of microbial cell debris allowed for NFRTC processing at significantly reduced polymer content, relative to pure PHB-based NFRTCs. Results further indicate that current commercial PHB production yields are sufficiently high to produce composites comparable to those manufactured with purified PHB.