VTechWorks Repository :: Browsing by Author "Barone, Justin R."

Browsing by Author "Barone, Justin R."

Now showing 1 - 20 of 46

Advancing characterization techniques for structure-property determination of in-situ lignocelluloses
Chowdhury, Sudip (Virginia Tech, 2011-07-21)
The global progression towards sustainable energy, materials and chemicals requires novel and improved analytical tools to understand and optimize lignocellulosic biomass utilization. In an effort to advance lignocellulose characterization, gain insights into biomass processing, and obtain novel perspectives on cell wall ultrastructure, this study utilizes three principal polymer characterization techniques, namely compressive-torsion dynamic mechanical analysis (DMA), deuterium quadrupolar nuclear magnetic resonance (2H NMR) and rheo-infrared spectroscopy. A novel parallel-plate compressive-torsion DMA protocol is developed to analyze very small solvent-plasticized biomass specimens with or without mechanical integrity. The benefits and limitations of this technique are demonstrated by comparing it to a conventional tensile-torsion DMA while analyzing various solvent-plasticized lignocelluloses. The rheology of wood in various organic solvents is studied through dynamic thermal scans, Time/temperature superposition (TTS) and fragility analysis. Plasticizing solvents and wood grain orientation significantly affected the lignin glass-transition temperature. Dynamic TTS reveals that while all storage modulus data shift smoothly, the thermorheological complexity of solvent-plasticized wood becomes evident in loss component master curves. It is argued that the plasticized lignocellulose TTS is insightful and potentially useful, although it fails to satisfy the classic TTS validity criteria. Subsequently, it is justified that the fragility analysis is a better suited treatment than the WLF model to investigate cooperative segmental motions of plasticized wood. Deuterium quadrupolar NMR reveals a new perspective on the orientation of amorphous wood polymers and two distinct amorphous polymer domains: a highly oriented phase in the S2 layer of the secondary cell wall and an isotropic phase postulated to occur in the compound middle lamella (CML). If the origin of the isotropic phase is confirmed to arise from the CML, then this technique provides a way to independently investigate the morphology and phase dynamics of CML and S2 in an intact tissue, and should bring novel insights into deconstructive strategies specific to the oriented and unoriented domains. Finally the effects of a wood-adhesion promoter (hydroxymethyl resorcinol, HMR) on in-situ wood polymers are studied to elucidate the still unresolved HMR-lignocellulose interactions. DMA, creep-TTS and 2H NMR reveal that HMR increases the crosslink density and restricts the mobility of wood amorphous phase. Rheo-IR spectroscopy shows that the molecular stress-transfer mechanism is altered within the wood cell wall.
Antibody Purification from Tobacco by Protein A Affinity Chromatography
Hey, Carolyn McKenzie (Virginia Tech, 2010-04-29)
Antibodies represent the largest group of biopharmaceuticals. Due to the nature of their clinical applications, they often need to be produced in large quantities. Plants have distinct advantages of producing large quantities of recombinant proteins, and tobacco is arguably the most promising plant for plant-made-pharmaceuticals (PMP) due to its high biomass yields and robust transformation technology. However, to produce proteins using transgenic tobacco for human applications, purification of the proteins is challenging. On the other hand, Protein A, a bacterial cell wall protein isolated from Staphylococcus aureus that binds to the Fc regions of immunoglobulins, is useful to the isolation and purification of antibodies. An affinity chromatography purification step utilizing Protein A resin introduced early in the purification process can reduce successive unit operations, thereby reducing the overall process cost. However, directly applying tobacco extract to Protein A chromatography columns may be problematic due to the non-specific binding of native tobacco proteins (NTP). In this project, three different Protein A resins, ProSepvA High Capacity, ProSep-vA Ultra, and ProSep Ultra Plus, marketed by Millipore, were studied to provide valuable information for future downstream processes for antibody purification from transgenic tobacco. The efficiency of the post load wash buffer to reduce non-specific binding of NTP to the ProSep A resins were evaluated by altering the ionic strength and pH. Lower salt concentrations of sodium chloride (NaCl) in the post load wash preformed best at reducing the non-specific binding of NTP to the ProSep A resins, while higher salt concentrations were more effective at reducing the amount of NTP contaminants present during elution of the columns. Using a post load wash buffer with an intermediate pH between the binding buffer and the elution buffer was more efficient at eluting our model antibody, human IgG. However, lowering the ionic strength and the pH of the post load wash buffer resulted in a greater presence of IgG prematurely eluting from the ProSep A resins. The non-specific binding of NTP to the resins reduced the dynamic binding capacity (DBC) of the resins after repeated cycles of tobacco extract samples were loaded onto the column. Nevertheless, cleaning the columns with denaturing solutions, such as urea or guanidine hydrochloride, every 8-10 cycles was effective in regenerating the DBC of the resins and prolonging the life cycle of the resins. This is important to evaluating the economic feasibility of directly using Protein A chromatography to recover antibodies from tobacco extract. Of the three Protein A resins studied, ProSep Ultra Plus performed best for antibody purification from tobacco using a PBS wash buffer with a lower ionic strength of 140mM NaCl and an intermediate pH of 5.
Application of Functional Amyloids in Morphological Control and in Self-assembled Composites
Claunch, Elizabeth Carson (Virginia Tech, 2013-05-14)
Amyloids are self-assembled protein materials containing beta-sheets. While most studies focus on amyloids as the pathogen in neurodegenerative disease, there are instances of "functional" amyloids used to preserve life. Functional amyloids serve as an inspiration in materials design. In this study, it is shown that wheat gluten (WG) and gliadin:myoglobin (Gd:My) amyloid morphology can be varied from predominantly fibrillar at low polypeptide concentration to predominantly globular at high polypeptide concentration as measured at the nanometer scale using atomic force microscopy (AFM). The ability to control the morphology of a material allows control of its properties. Fourier transform infrared (FTIR) spectroscopy shows that at low concentration, fibrils require interdigitation of methyl groups on alanine (A), isoleucine (I), leucine (L), and valine (V). At higher concentration, globules do not have the same interdigitation of methyl groups but more random hydrophobic interactions. The concentration dependence of the morphology is shown as a kinetic effect where many polypeptides aggregate very quickly through hydrophobic interactions to produce globules while smaller populations of polypeptides aggregate slowly through well-defined hydrophobic interactions to form fibrils. Functional amyloids also provide a means of creating a low energy process for composites. Poor fiber/matrix bonding and processing degradation have been observed in previous WG based composites. This study aims to improve upon these flaws by implementing a self-assembly process to fabricate self-reinforced wheat gluten composites. These composites are processed in aqueous solution at neutral pH by allowing the fibers to form in a matrix of unassembled peptides. The fiber and the matrix are formed from the same solution, thus the two components create a compatible system with ideal interfacial interaction for a composite. The fibers in the composite are about 10 microns in diameter and can be several millimeters long. It has been observed that the number of fibers present along the fracture surface influences the modulus of the composite. In this study, self-assembled wheat gluten composites are formed and then characterized with 3-point bend (3PB) mechanical testing, scanning electron microscopy (SEM), and Fourier transform infrared (FTIR) spectroscopy.
Bio-based composites that mimic the plant cell wall
Li, Zhuo (Virginia Tech, 2009-04-22)
Nature creates high performance materials under modest conditions, i.e., neutral pH and ambient temperature and pressure. One of the most significant materials is the plant cell wall. The plant cell wall is a composite of oriented cellulose microfibrils reinforcing a lignin/hemicellulose matrix. In principle, the plant cell wall composite is designed much like a synthetic fiber-reinforced polymer composite. Unlike synthetic composites, the plant cell wall has an excellent combination of high modulus, strength, toughness and low density that originates in the optimal interactions between the biopolymers. Therefore, to produce high performance composites, a unique route may be to mimic a biological system like the plant cell wall. The present work focuses on understanding the thermodynamics of biopolymer assembly to exploit the process in vitro. In our system, we use an already polymerized nanocellulose template and polymerize phenolic monomers on the template using a peroxidase enzyme. In the first part, we have polymerized phenol using horseradish peroxidase (HRP) in the presence of TEMPO-oxidized nanocellulose. Similar to native plant cell wall structures, the polyphenol-nanocellulose composite had intimate mixing of polyphenol and cellulose at the nanoscale with the presence of cellulose promoting a uniquely organized structure. The obtained composite material showed synergy that enhanced the thermal stability, hydrophobicity, and possibly mechanical properties. In the second part, monolignol coniferyl alcohol was polymerized in the presence of nanocellulose by the same procedure. A comparison between the polyphenol composite and poly(coniferyl alcohol) (PCA) composite revealed that the propanyl substitution imparted flexibility to the PCA molecules so that they could bend and form a hollow globule structure to envelope nanocellulose inside. Polyphenol could not do this because of its rigidity.
Biopolymer Structure Analysis and Saccharification of Glycerol Thermal Processed Biomass
Zhang, Wei (Virginia Tech, 2015-01-31)
Glycerol thermal processing (GTP) is studied as a novel biomass pretreatment method in this research with the purposes to facilitate biopolymer fractionation and biomass saccharification. This approach is performed by treating sweet gum particles on polymer processing equipment at high temperatures and short times in the presence of anhydrous glycerol. Nine severity conditions are studied to assess the impact of time and temperature during the processing on biopolymer structure and conversion. The GTP pretreatment results in the disruption of cell wall networks by increasing the removal of side-chain sugars and lignin-carbohydrate linkages based on severity conditions. After pretreatment, 41% of the lignin and 68% of the xylan is recovered in a dry powdered form by subsequent extractions without additional catalysts, leaving a relatively pure cellulose fraction, 84% glucan, as found in chemical pulps. Lignin structural analysis indicated GTP processing resulted in extensive degradation of B-aryl ether bonds through the C-y elimination, followed by abundant phenolic hydroxyl liberation. At the same time, condensation occurred in the GTP lignin, providing relatively high molecular weight, near to that of the enzymatic mild acidolysis lignin. Better thermal stability was observed for this GTP lignin. In addition to lignin, xylan was successfully isolated as another polymer stream after GTP pretreatment. The recovered water insoluble xylan (WIX) was predominant alkali soluble fraction with a maximum purity of 84% and comparable molecular weight to xylan isolated from non-pretreated fibers. Additionally, the narrow molecular weight distribution of recovered WIX, was arisen from the pre-extraction of low molecular weight water-soluble xylan. Additionally, a 20-fold increase of the ultimate enzymatic saccharification for GTP pretreated biomass was observed even with significant amounts of lignin and xylan remaining on the non-extracted fiber. The shear and heat processing caused a disintegrated cell wall structure with formation of biomass debris and release of cellulose fibrils, enhancing surface area and most likely porosity. These structural changes were responsible for the improved biomass digestibility. Additionally, no significant inhibitory compounds for saccharification are produced during GTP processing, even at high temperatures. While lignin extraction did not promote improvement in hydrolysis rates, further xylan extraction greatly increases the initial enzymatic hydrolysis rate and final level of saccharification. The serial of studies fully demonstrate glycerol thermal processing as a novel pretreatment method to enhance biomass saccharification for biofuel production, as well as facilitate biopolymer fractionation. Moreover, the study shows the impact of thermally introduced structural changes to wood biopolymers when heated in anhydrous environments in the presence of hydrogen bonding solvent.
Catalytic Pyrolysis of Cellulose, Hemicellulose and Lignin Model Compounds
Atadana, Frederick Williams (Virginia Tech, 2010-01-25)
The effect of HZSM-5 catalyst and NaOH pretreatment on the product distribution and bio oil properties from pyrolysis of cellulose, hemicellulose and lignin model compounds was investigated at 450 °C. NaOH pretreated and untreated cellulose was pyrolyzed on sand and the HZSM-5 catalysts; VPISU001 HZSM-5, BASF HZSM-5, and Sud-Chemie HZSM-5. The pyrolysis of cellulose on BASF and Sud-Chemie HZSM-5 catalysts increased the yields of the organic liquid fraction, total liquid and char while decreasing the gas yields. However the catalyst decreased the organic and char yields while increasing the water yields but there was no change in gas yields. The NaOH treatment caused a decrease in the organic and total oil yields relative to the control but the char yield increased. The change in gas yields was not significant. The characterization of the oils using FTIR and ¹³C−nmr showed that, the VPISU001 HZSM-5 with and without NaOH pretreatment caused elimination of the levoglucosan fraction while increasing the aromatic fraction. The NaOH pretreated cellulose pyrolyzed on sand reduced the levoglucosan groups while increasing the aromatic fraction of the bio oil. In the hemicellulose studies, birchwood xylan and NaOH treated xylan samples were pyrolyzed on sand and VPISU001 HZSM-5 catalyst. The organic liquid yields were very low and ranged from 3.3 wt% to 7.2 wt%, the water yields ranged from 17.8-25.7 wt%, the char yield were 17.8-25 wt% and gas yield were 40.9-49.6 wt%. The HZSM-5 catalysts increased the water and gas yields and produced the lowest char yield. NaOH pretreatment produced the lowest water yield while the char yield was the highest. The combined effect of NaOH pretreatment and HZSM-5 produced the lowest organic yield and highest char yield. The FTIR and ¹³C-nmr analyses of the organic liquids showed that the HZSM-5 catalyst promoted the formation of aromatic products, while the NaOH pretreatment promoted the formation of aliphatic hydrocarbons. The combined effect of NaOH pretreatment and HZSM-5 catalyst seem to promote the formation of anhydrosugars. The main gases evolved were CO, CO₂ and low molecular weight hydrocarbons. The HZSM-5 catalyst promoted CO formation while NaOH pretreatment promoted CO₂. The HZSM-5 catalyst produced the highest yield of low molecular weight hydrocarbon gases. The lignin and model compounds studies involved using low molecular weight kraft lignin, guaiacol, and syringol which were pyrolyzed on sand and VPISU001 HZSM-5 catalyst at 450 °C. The kraft lignin pyrolysis produced low liquid and gas yields and high char yields. The HZSM-5 catalysts increased the water yield and decreased the organic liquid yield. NaOH pretreatment increased the char yield and decreased the liquid products. NaOH and the HZSM-5 catalyst together decreased the char and increased the gas yields. The ¹³C-nmr and FTIR analysis showed that NaOH pretreatment promoted the formation of mainly guaiacol while the HZSM-5 catalyst formed different aromatic components. NaOH pretreatment promoted the formation of more CO₂ than CO whilst HZSM-5 catalyst promoted the formation of more CO than CO₂. Methane formation was enhanced by NaOH pretreatment. Other hydrocarbon gases were however enhanced by the HZSM-5 catalysts. Pyrolysis of the model compounds on the HSZM-5 catalyst showed an increase in pyrolytic water. The HZSM-5 catalyst promoted demethylation in syringol pyrolysis as compared to guaiacol.
Cell-Free Biosystems Comprised of Synthetic Enzymatic Pathways: Development of Building Blocks, Immobilization of Enzymes, Stabilization of Cascade Enzymes, and Generation of Hydrogen
Myung, Suwan (Virginia Tech, 2013-05-08)
The production of hydrogen from low-cost abundant renewable biomass would be vital to sustainable development. Cell-free (in vitro) biosystems comprised of synthetic enzymatic pathways would be a promising biomanufacturing platform due to several advantages, such as high product yield, fast reaction rate, easy control and access, and so on. However, it is essential to produce (purified) enzymes at low costs and stabilize them for long periods to decrease biocatalyst costs. Thermophilic recombinant enzymes as building blocks were discovered and developed: fructose 1,6-bisphosphatase (FBP) from Thermotoga maritime, phosphoglucose isomerase (PGI) from Clostridium thermocellum, triose phosphate isomerase (TIM) from Thermus thermophiles and fructose bisphosphate aldolase (ALD) from T. maritima and T. thermophilus. The recombinant proteins were over-expressed in E. coli, purified and characterized. For purification and stabilization of enzymes, one-step, simple, low-cost purification and immobilization methods were developed based on simple adsorption of cellulose-binding module (CBM)-tagged protein on the external surface of high-capacity regenerated amorphous cellulose. Also, a simple, low-cost purification method of thermophilic enzymes was developed utilizing a combination of heat and ammonium sulfate precipitation. For development of cascade enzymes as building modules (biocatalyst modules), it was discovered that the presence of other enzymes/proteins had a strong synergetic effect on the stabilization of the thermolabile enzyme (e.g., PGI) due to the in vitro macromolecular crowding effect. And substrate channeling among CBM-tagged self-assembled three-enzyme complex (synthetic matabolon) immobilized on the easily-recycled cellulose-containing magnetic nanoparticles can not only increase cascade reaction rates greatly, but also decrease enzyme cost in cell-free biosystems. The high product yield and fast reaction rate of dihydrogen from sucrose was validated in a batch reaction containing fifteen enzymes comprising a non-natural synthetic pathway. The yield of dihydrogen production from 2 mM of sucrose was 96.7 % compared to theoretical yield at 37 °C. The maximum rate was increased 3.1 fold when the substrate concentration was increased from 2 to 50 mM in a fed-batch reaction. The research and development of cell-free biosystems for biomanufacturing require more efforts, especially in low-cost recombinant thermostable enzymes as building blocks, efficient cofactor recycling, enzyme and cofactor stabilization, and fast reaction rates.
Characterization of a Novel Biodegradable Material to Reduce Emission of Ammonia
Adjei, Thomas (Virginia Tech, 2007-12-06)
A novel biodegradable ammonia control material was developed from steam exploded corn cob and its adsorption capacity was studied by packed column and titration method. The packed column studies showed that the maximum absorption capacities of the raw corn cob (RCC) and the steam exploded corn cob (SECC) were 10.45 mg NHB3B/gRCC and 59.80 mg NHB3B/gSECC respectively. However, the titration of the water slurries with a NHB4BOH showed that the capacity of the SECC was 14.4 times that of RCC. The large difference between the packed column (SECC/RCC = 5.7) and the slurry titration (SECC/RCC = 14.4) was probably because: (1) the initial ammonia reaction products blocked the pores of the SECC and reduced diffusion into the pore structure; (2) the ammonia gas flow rates were too high and therefore the gas did not penetrate the pores; (3) the gas contact time was below the equilibrium value; and (4) since interior pore surface area is usually larger than the external surface area of a particle, it appears the low column SECC/RCC ratio is due to reactions on the SECC particle surface whereas the slurry result was a combination of both. Fourier Transform Infrared, FTIR spectroscopy was conducted on RCC, SECC, ammonia adsorbed on RCC and ammonia adsorbed on SECC in the range 4000–700 cmP-1P. The FTIR bands in the region between 1500 and 2000 cmPâ 1P showed a considerable difference between RCC and SECC. When SECC was treated with ammonia, the carboxylic functional group peak at 1700 cmP-1P was reduced and a new peak was observed at 1584 cmP-1P. The adsorption, desorption test and the heat of adsorption results suggested combined physisorption and chemisorption of ammonia on SECC but chemisorption was found to play an important role in ammonia removal. The BET specific surface area of RCC was 3.4 m2/g whilst that SECC was less than 1 m2/g. Although SECC had a low surface area compared with RCC its adsorption capacity was found to be greater than that of RCC meaning the adsorption process is chemically controlled. Also, the pore size distribution showed that RCC exhibited both macroporosity and mesoporosity whilst SECC showed only mesoporosity. It is interesting to note that upon steam exploding RCC, the macropores within RCC collapsed to form more mesopores in SECC. The high uptake of SECC was determined to be its small pore width compared to that RCC. Simultaneous Differential Scanning Calorimetry, DSC and Thermal Gravimetric Analyzer, TGA, was used to determine the heat of adsorption of ammonia on SECC. The heat of adsorption of ammonia on SECC was 33.00 kJ per mole of NHB3B. This study shows that SECC could be potentially used to remove NHB3B from various emission sources.
Coenzyme engineering of NAD(P)+ dependent dehydrogenases
Huang, Rui (Virginia Tech, 2017-12-11)
Coenzyme nicotinamide adenine dinucleotide (NAD, including the oxidized form-- NAD+ and reduced form--NADH) and the phosphorylated form--nicotinamide adenine dinucleotide phosphate (NADP, including NADP+ and NADPH) are two of the most important biological electron carriers. Most NAD(P) dependent redox enzymes show a preference of either NADP or NAD as an electron acceptor or donor depending on their unique metabolic roles. In biocatalysis, the low enzymatic activities with unnatural coenzymes have made it difficult to replace costly NADP with economically advantageous NAD or other biomimetic coenzyme for catalysis. This is a significant challenge that must be addressed should in vitro biocatalysis be a viable option for the practical production of low-value biocommodities (i.e., biohydrogen). There is a significant need to first address the coenzyme selectivity of the NADP-dependent dehydrogenases and evolve mutated enzymes that accept biomimetic coenzymes. This is a major focus of this dissertation. Establishment of efficient screening methods to identify beneficial mutants from an enzymatic library is the most challenging task of coenzyme engineering of dehydrogenases. To fine tune the coenzyme preference of dehydrogenases to allow economical hydrogen production, we developed a double-layer Petri-dish based screening method to identify positive mutant of the Moorella thermoacetica 6PGDH (Moth6PGDH) with a more than 4,278-fold reversal of coenzyme selectivity from NADP+ to NAD+. This method was also used to screen the thermostable mutant of a highly active glucose 6-phosphate dehydrogenase from the mesophilic host Zymomonas mobilis. The resulting best mutant Mut 4-1 showed a more than 124-fold improvement of half-life times at 60oC without compromising the specific activity. The screening method was further upgraded for the coenzyme engineering of Thermotaga maritima 6PGDH (Tm6PGDH) on the biomimetic coenzyme NMN+. Through six-rounds of directed evolution and screening, the best mutant showed a more than 50-fold improvement in catalytic efficiency on NMN+ and a more than 6-fold increased hydrogen productivity rate from 6-phosphogluconate and NMN+ compared to those of wild-type enzyme. Together, these results demonstrated the effectiveness of screening methods developed in this research for coenzyme engineering of NAD(P) dependent dehydrogenase and efficient use of the less costly coenzyme in ivSB based hydrogen production.
Consistent Fabrication of Ultrasmall PLGA Nanoparticles and their Potential Biomedical Applications
Lohneis, Taylor Paige (Virginia Tech, 2019-12-04)
Nanotechnology and its potential for biomedical applications has become an area of increasing interest over the last few decades. Specifically, ultrasmall nanoparticles, ranging in size from 5 to 50 nm, are highly sought after for their physical and chemical properties and their ability to be easily transmitted though the bloodstream. By adjusting the material properties, size, surface potential, morphology, surface modifications, and more, of nanoparticles, it is possible to tailor them to a specific use in biomedical areas such as drug and gene delivery, biodetection of pathogens or proteins, and tissue engineering. The aim of this study was to fabricate ultrasmall poly-(lactic-co-glycolic acid) nanoparticles (PLGA NPs) using a quick and easy nanoprecipitation method1, with some modifications, for general use in various biomedical areas. Nanoprecipitation of two solutions – PLGA dissolved in acetonitrile and aqueous poly(vinyl alcohol) (PVA) – at varying concentrations produced ultrasmall nanoparticles that range in size, on average, from 10 to 30 nm. By the data collected from this study, a selection method can be used to choose a desired PLGA nanoparticle size given a potential biomedical application. The desired nanoparticle can be fabricated using specific concentrations of the two nanoprecipitation solutions. Size of the ultrasmall PLGA NPs was characterized by dynamic light scattering (DLS) and confirmed by transmission electron microscopy (TEM). Spherical morphology of the PLGA NPs was also proved by TEM. By generalizing the ultrasmall PLGA NP fabrication process, the idea is that these NPs will be able to be used in various biomedical applications depending on the goal of the furthered study. As an example of potential application, ~15 to 20 nm PLGA NPs were consistently fabricated for use as virus-like particle (VLP) scaffolds. Following formation, PLGA NPs were introduced to modified human papillomavirus (HPV) protein during protein refolding and assembly into virus-like particles (VLPs) via buffer exchange. The size of the VLPs was monitored with and without PLGA nanoparticles present in solution during the refolding process and TEM images were collected to confirm encapsulation.
Controlled Hybrid Material Synthesis using Synthetic Biology
Scott, Felicia Yi Xia (Virginia Tech, 2017-06-02)
The concept of creating a hybrid material is motivated by the development of an improved product with acquired properties by amalgamation of components with specific desirable traits. These new attributes can range from improvements upon existing properties, such as strength and durability, to the acquisition of new abilities, such as magnetism and conductivity. Currently, the concept of an organic-inorganic hybrid material typically describes the integration of an inorganic polymer with organically derived proteins. By building on this idea and applying the advanced technologies available today, it is possible to combine living and nonliving components to synthesize functional materials possessing unique abilities of living cells such as self-healing, evolvability, and adaptability. Furthermore, artificial gene regulation, achievable through synthetic biology, allows for an additional dimension of the control of hybrid material function. Here, I genetically engineer E. coli with a tightly controlled artificial protein construct, allowing for inducible expression of different amounts of the surface anchored protein by addition of varying concentrations of L-arabinose. The presence of the surface protein allows the cells to bind nonliving nanoparticle substrates, effectively turning the cells into living crosslinkers. By using the living crosslinker, I was able to successfully synthesize a robust, macroscale living-nonliving hybrid material with magnetic characteristics. Furthermore, by varying the particle size and inducer concentration, the resulting material exhibited alterations in structure and function. Finally, I was able to manipulate material kinetics within a PDMS channel by applying fluctuating magnetic fields and demonstrate material durability. These results demonstrate the ability to manipulate synthesis of living-nonliving hybrid materials, which demonstrate the potential for use in promising applications in areas such as environmental monitoring and micromachining. Additionally, this work serves as a foundational step toward the integration of synthetic biology with tissue engineering by exploiting the possibility of controlling material properties with genetic engineering.
Creation of Ovalbumin Based Scaffolds for Bone Tissue Regeneration
Farrar, Gabrielle (Virginia Tech, 2009-04-24)
Bio-based materials are a viable alternative to synthetic materials for tissue engineering. Although many bio-based materials have been used, Ovalbumin (OA) has not yet been researched to create 3D structures that promote cellular responses. Micro-porous scaffolds are a promising construct for bone tissue regeneration; therefore OA crosslinked with three different concentrations (10%, 15% and 20%) of glutaraldehyde (GA) was used in this research. After fabrication, a porous morphology was observed using SEM. Average pore sizes were found to be comparable to scaffolds previously shown to promote cellular response. A TNBS assay determined percent crosslinking in the scaffolds, however there was no significant difference in percent crosslinking despite differing GA concentrations used. Possible explanations include an excess of GA was used. Using DSC, a glass transition temperature (Tg) was found for control indicating the scaffolds are amorphous. Average dry and wet compressive strengths were also found. As expected, differing GA concentrations had no significant effect on Tg and average compressive strengths due to an excess used. Scaffolds were mechanically tested at 37°C with no significant difference found; therefore these scaffolds can be used in the body. It was shown through cell studies that MC3T3-E1 pre-osteoblast cells significantly increased in number on the 10% and 15% scaffolds, therefore cell proliferation occurred. Because of a positive cellular response, 10% GA scaffolds were used for differentiation studies that showed an increase in osteocalcin at 21 days and alkaline phosphatase levels for scaffolds cultured for 14 days. Overall OA scaffolds have shown to be a promising 3D construct for bone tissue regeneration.
Crossing the Air-Water Interface: Inspiration from Nature
Chang, Brian Lida (Virginia Tech, 2018-06-01)
This dissertation aims to contribute toward the understanding of water-entry and -exit behaviors in nature. Since water is nearly a thousand times denser than air, transitioning between the two mediums is often associated with significant changes in force. Three topics with implications in water-entry are discussed, along with a fourth topic on water-exit. For a plunge-diving seabird, the first two stages of water-entry (initial impact and air-cavity formation) create large stresses on the bird's neck. Linear stability analysis of a cone-beam system impacting water shows buckling and non-buckling behaviors on the beam, which is extended to the diving birds. The next topic is related to the third stage of water-entry (air-cavity pinch-off), in which the chest feathers come in contact with the water. Here, the elasticity of Northern Gannet contour feathers is calculated using the nonlinear bending equation. The third topic will explore the formation of ripples along air cavity walls and their resulting force after pinch-off. An acoustic model predicts the observed wavelengths of the ripples. The final topic will delve into the mechanics of how animals leap out of water. A scaling law that balances the power of thrust and drag will predict the height of the jump. Finally, a bio-inspired robot was built to help identify physical conditions required to jump out of water.
Degenerate oligonucleotide primed amplification of genomic DNA for combinatorial screening libraries and strain enrichment
Freedman, Benjamin Gordon (Virginia Tech, 2014-12-22)
Combinatorial approaches in metabolic engineering can make use of randomized mutations and/or overexpression of randomized DNA fragments. When DNA fragments are obtained from a common genome or metagenome and packaged into the same expression vector, this is referred to as a DNA library. Generating quality DNA libraries that incorporate broad genetic diversity is challenging, despite the availability of published protocols. In response, a novel, efficient, and reproducible technique for creating DNA libraries was created in this research based on whole genome amplification using degenerate oligonucleotide primed PCR (DOP-PCR). The approach can produce DNA libraries from nanograms of a template genome or the metagenome of multiple microbial populations. The DOP-PCR primers contain random bases, and thermodynamics of hairpin formation was used to design primers capable of binding randomly to template DNA for amplification with minimal bias. Next-generation high-throughput sequencing was used to determine the design is capable of amplifying up to 98% of template genomic DNA and consistently out-performed other DOP-PCR primers. Application of these new DOP-PCR amplified DNA libraries was demonstrated in multiple strain enrichments to isolate genetic library fragments capable of (i) increasing tolerance of E. coli ER2256 to toxic levels of 1-butanol by doubling the growth rate of the culture, (ii) redirecting metabolism to ethanol and pyruvate production (over 250% increase in yield) in Clostridium cellulolyticum when consuming cellobiose, and (iii) enhancing L-arginine production when used in conjunction with a new synthetic gene circuit.
Design and Construction of Large Amyloid Fibers
Ridgley, Devin M.; Rippner, Caitlin M. W.; Barone, Justin R. (MDPI, 2015-04-16)
Mixtures of “template” and “adder” proteins self-assemble into large amyloid fibers of varying morphology and modulus. Fibers range from low modulus, rectangular cross-sectioned tapes to high modulus, circular cross-sectioned cylinders. Varying the proteins in the mixture can elicit “in-between” morphologies, such as elliptical cross-sectioned fibers and twisted tapes, both of which have moduli in-between rectangular tapes and cylindrical fibers. Experiments on mixtures of proteins of known amino acid sequence show that control of the large amyloid fiber morphology is dependent on the amount of glutamine repeats or “Q-blocks” relative to hydrophobic side chained amino acids such as alanine, isoleucine, leucine, and valine in the adder protein. Adder proteins with only hydrophobic groups form low modulus rectangular cross-sections and increasing the Q-block content allows excess hydrogen bonding on amide groups that results in twist and higher modulus. The experimental results show that large amyloid fibers of specific shape and modulus can be designed and controlled at the molecular level.
Development of subunit vaccines against porcine reproductive and respiratory syndrome virus (PRRSV)
Hu, Jianzhong (Virginia Tech, 2012-08-06)
Since emerging in Europe and the US, PRRS has spread globally and become the most significant infectious disease currently devastating the swine industry. In the US alone, the economic losses caused by this disease amount to more than 560 million US dollars every year. Modified-live PRRSV vaccines (MLV) are the most effective option currently available for the control of the disease. MLVs can confer solid protection against homologous re-infection and have significant effects in reducing viral shedding. But the vaccine efficacy varies upon heterologous challenge. None of the current vaccines are able to completely prevent respiratory infection, transplacental transmission, as well as pig-to-pig transmission of the virus. More importantly, the intrinsic risk of MLV vaccine to revert to virulent virus under farm conditions poses a great safety concern. The unsatisfactory efficacy and safety of current PRRSV vaccines drives the continuous efforts of developing a new generation of vaccines. The strategy we focus on for novel PRRSV vaccine development is subunit vaccine. The reasons for choosing this strategy are: 1) subunit vaccines only contain the immunogenic fragments of a pathogen. Administration of such pathogen fragments eliminates the risk of pathogens reverting back to their virulent form as in the case of modified live vaccines. 2) Subunit vaccines have advantages in terms of vaccine production since a well-defined pathogen fragment can more easily be produced consistently. To achieve of our goal of developing safe and efficacious subunit vaccines against PRRSV, three projects were completed. First, a scalable process for purification of PRRSV particles from cell culture was developed. This process produced purified viral particles for ELISA and cell-based assays used in vaccine development. Second, a plant-made oral subunit vaccine against PRRSV was developed. Administration of the plant-made vaccine, the vaccinated animals produced virus-specific serum and intestine mucosal antibodies with neutralization activity, as well as cellular immune responses with a preference of virus-specific IFN-γ production. Since neutralization antibodies and virus-specific IFN-γ response are the crucial factors contributing to protection against PRRSV infection, the plant-made oral subunit vaccine strategy is an attractive strategy for developing a new generation of the vaccine to control PRRS disease. Third, a chimeric protein consisting of the ectodomains of viral M and GP5 proteins was expressed and purified. The protein product showed a single band on a silver-stained gel and contained an endotoxin level of less than 10 EU/mg protein. In addition, the purified protein showed expected bioactivities. It was antigenic, could bind to a cellular receptor for the virus (heparan sulfate), and could block virus infection of susceptible cells. Therefore, the chimeric protein is a promising subunit vaccine candidate against PRRSV.
Direct conversion of chemical energy to mechanical work using a phosphate charged protein
Shen, Ying (Virginia Tech, 2010-04-09)
Nature is able to convert chemical energy into mechanical work under modest conditions, i.e., physiological pH and ambient temperature and pressure. One of the most interesting systems is muscle modeled as the "sliding filament" system. The sliding filament system is a combination of a thin actin filament and a thick myosin filament that slide over one another by breaking the "energy-rich" pyrophosphate bond of ATP. The energy from ATP hydrolysis is used for mechanical motion and the energy lost during this process is used to heat our body. In biology, the sliding filament system is taken as a fairly effective model. For engineering systems, the energy lost to heat needs to be reduced to build an efficient energy converter. In our research, we use a phosphate charged protein, casein, and react it with divinyl sulfone (DVS) through a Michael addition reaction to produce a cross-linked gel. The protein gel could be ephosphorylated at standard conditions using bovine phosphatase (bp) and re-phosporylated using casein kinase. When attached to the protein, the negatively charged phosphate groups cause the gel to expand from repulsion. When removed, the protein contracts. Therefore, work is realized without sliding friction, which is the origin of the large energy loss in muscle. FT-IR spectroscopy allows us to follow the two biochemical reactions. We also show a thermodynamic analysis of the work and offer an estimation of the most basic term.
Editorial [Green Materials]
Barone, Justin R. (ICE Publishing, 2021-12-01)
Effect of de novo peptide properties on self-assembling large amyloid fibers
Rippner, Caitlin Marie Weigand (Virginia Tech, 2013-05-14)
Amyloid aggregation involves the spontaneous formation of fibers from misfolded proteins. This process requires low energy input, results in robust fibers, and is thus of interest from a materials manufacturing perspective. The effect of glutamine content and hydrophobicity of template peptides on amyloid aggregation of a template-peptide system involving myoglobin was studied at near-physiological conditions by Fourier transform infrared spectroscopy, atomic force microscopy, field emission scanning electron microscopy, and nanoindentation. Hydrophobic interactions were found to be important for controlled hierarchical fiber growth via a cooperative mechanism, with the largest effect in myoglobin mixtures. Hydrophobic packing increased for most systems as aggregation progressed. The largest changes in structure occurred upon drying. When myoglobin was present with the highest glutamine-containing template (P7), the high glutamine peptide was not effective as a template, since it appeared to prefer self-catalysis. A low level of glutamine in some unordered templates was insufficient for amyloid development. However, templating was more important in glutamine-free templates mixed with myoglobin, which formed fibers with a surprisingly high elastic modulus. This may have been due to template patterning. Nanoindentation results confirmed that glutamine blocks were not necessary for strong intermolecular interactions and cooperative fibril formation.
Effects of Thermomechanical Refining on Douglas fir Wood
Tasooji, Mohammad (Virginia Tech, 2018-07-03)
Medium density fiberboard (MDF) production uses thermomechanically refined fiber processed under shear with high pressure steam. The industry evaluates fiber quality with visual and tactile inspection, emphasizing fiber dimensions, morphology, and bulk density. Considering wood reactivity, the hypothesis is that a variety of chemical and physical changes must occur that are not apparent in visual/tactile inspection. An industry/university cooperation, this work studies effects of refining energy (adjusted by refiner-plate gap) on fiber: size, porosity, surface area, surface and bulk chemistry, fiber crystallinity and rheology, and fiber interaction with amino resins. The intention is to reveal novel aspects of fiber quality that might impact MDF properties or process control efficiency, specific to a single industrial facility. In cooperation with a North American MDF Douglas fir plant, two refining energies were used to produce resin and additive-free fibers. Refining reduced fiber dimensions and increased bulk density, more so at the highest energy. Thermoporosimetry showed increases in sub-micron scale porosity, greatest at the highest energy. Mercury intrusion porosimetry (MIP) revealed porosity changes on a higher dimensional scale. Brunauer-Emmett-Teller gas adsorption and MIP showed that refining increased specific surface area, more so at the highest energy. Inverse gas chromatography showed that the lowest refining energy produced surfaces dominated by lignin and/or extractives. The highest energy produced more fiber damage, revealing higher energy active sites. A novel rheological method was devised to study fiber compaction and densification; it did not distinguish fiber types, but valuable aspects of mechano-sorption and densification were observed. Refining caused substantial polysaccharide degradation, and other degradative effects that sometimes correlated with higher refining energy. Lignin acidolysis was detected using nitrobenzene oxidation, conductometric titration of free phenols, and formaldehyde determination. Formaldehyde was generated via the C2 lignin acidolysis pathway, but C3 cleavage was the dominant lignin reaction. Observations suggested that in-line formaldehyde monitoring might be useful for process control during biomass processing. According to rheological and thermogravimetric analysis, lignin acidolysis was not accompanied by repolymerization and crosslinking. Lignin repolymerization must have been prevented by the reaction of benzyl cations with non-lignin nucleophiles. This raises consideration of additives that compete for lignin benzyl cations, perhaps to promote lignin crosslinking and/or augment the lignin network with structures that impart useful properties. Fiber/amino resin interactions were studied with differential scanning calorimetry (DSC) and X-ray diffraction (XRD). All fiber types, refined and unrefined, caused only a slight increase in melamine-urea-formaldehyde (MUF) resin reactivity. Generally, all fiber types decreased the enthalpy of MUF cure, suggesting fiber absorption of small reactive species. But DSC did not reveal any dependency on fiber refining energy. According to XRD, all fiber types reduced crystallinity in cured MUF, more so with refined fiber, but independent of refining energy. The crystallinity in cured urea-formaldehyde resin was studied with one fiber type (highest refining energy); it caused a crystallinity decrease that was cure temperature dependent. This suggests that resin crystallinity could vary through the thickness of an MDF panel.

Browsing by Author "Barone, Justin R."

Results Per Page

Sort Options