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Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical

Lignocellulosic biomass represents a renewable domestic feedstock that can support large-scale biochemical production processes for fuels and specialty chemicals. However, cost-effective conversion of lignocellulosic sugars into valuable chemicals by microorganisms still remains a challenge. Biomass recalcitrance to saccharification, microbial substrate utilization, bioproduct titer toxicity, and toxic chemicals associated with chemical pretreatments are at the center of the bottlenecks limiting further commercialization of lignocellulose conversion. Genetic and metabolic engineering has allowed researchers to manipulate microorganisms to overcome some of these challenges, but new innovative approaches are needed to make the process more commercially viable. Transport proteins represent an underexplored target in genetic engineering that can potentially help to control the input of lignocellulosic substrate and output of products/toxins in microbial biocatalysts. In this work, I characterize and explore the use of transport systems to increase substrate utilization, conserve energy, increase tolerance, and enhance biocatalyst performance.
ContributorsKurgan, Gavin (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Misra, Rajeev (Committee member) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2018
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Description
Carbon dioxide (CO2) levels in the atmosphere have reached unprecedented levels due to increasing anthropogenic emissions and increasing energy demand. CO2 capture and utilization can aid in stabilizing atmospheric CO2 levels and producing carbon-neutral fuels. Utilizing hollow fiber membranes (HFMs) for microalgal cultivation accomplishes that via bubbleless gas-transfer,

Carbon dioxide (CO2) levels in the atmosphere have reached unprecedented levels due to increasing anthropogenic emissions and increasing energy demand. CO2 capture and utilization can aid in stabilizing atmospheric CO2 levels and producing carbon-neutral fuels. Utilizing hollow fiber membranes (HFMs) for microalgal cultivation accomplishes that via bubbleless gas-transfer, preventing CO2 loss to the atmosphere. Various lengths and geometries of HFMs were used to deliver CO2 to a sodium carbonate solution. A model was developed to calculate CO2 flux, mass-transfer coefficient (KL), and volumetric mass-transfer coefficient (KLa) based on carbonate equilibrium and the alkalinity of the solution. The model was also applied to a sparging system, whose performance was compared with that of the HFMs. Typically, HFMs are operated in closed-end mode or open-end mode. The former is characterized by a high transfer efficiency, while the latter provides the advantage of a high transfer rate. HFMs were evaluated for both modes of operation and a varying inlet CO2 concentration to determine the effect of inert gas and water vapor accumulation on transfer rates. For pure CO2, a closed-end module operated as efficiently as an open-end module. Closed-end modules perform significantly worse when CO2-enriched air was supplied. This was shown by the KLa values calculated using the model. Finally, a mass-balance model was constructed for the lumen of the membranes in order to provide insight into the gas-concentration profiles inside the fiber lumen. For dilute CO2 inlet streams, accumulation of inert gases -- nitrogen (N2), oxygen (O2), and water vapor (H2O) -- significantly affected module performance by reducing the average CO2 partial pressure in the membrane and diminishing the amount of interfacial mass-transfer area available for CO2 transfer.
ContributorsShesh, Tarun (Author) / Rittmann, Bruce E. (Thesis advisor) / Green, Matthew (Committee member) / Torres, Cesar (Committee member) / Arizona State University (Publisher)
Created2018
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Description
In our modern world the source of for many chemicals is to acquire and refine oil. This process is becoming an expensive to the environment and to human health. Alternative processes for acquiring the final product have been developed but still need work. One product that is valuable is butanol.

In our modern world the source of for many chemicals is to acquire and refine oil. This process is becoming an expensive to the environment and to human health. Alternative processes for acquiring the final product have been developed but still need work. One product that is valuable is butanol. The normal process for butanol production is very intensive but there is a method to produce butanol from bacteria. This process is better because it is more environmentally safe than using oil. One problem however is that when the bacteria produce too much butanol it reaches the toxicity limit and stops the production of butanol. In order to keep butanol from reaching the toxicity limit an adsorbent is used to remove the butanol without harming the bacteria. The adsorbent is a mesoporous carbon powder that allows the butanol to be adsorbed on it. This thesis explores different designs for a magnetic separation process to extract the carbon powder from the culture.
ContributorsChabra, Rohin (Author) / Nielsen, David (Thesis director) / Torres, Cesar (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor)
Created2015-05
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Description
The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is the use of nonribosomal peptide synthases (NRPSs). These are modular

The world today needs novel solutions to address current challenges in areas spanning areas from sustainable manufacturing to healthcare, and biotechnology offers the potential to help address some of these issues. One tool that offers opportunities across multiple industries is the use of nonribosomal peptide synthases (NRPSs). These are modular biological factories with individualized subunits that function in concert to create novel peptides.One element at the heart of environmental health debates today is plastics. Biodegradable alternatives for petroleum-based plastics is a necessity. One NRPS, cyanophycin synthetase (CphA), can produce cyanophycin grana protein (CGP), a polymer composed of a poly-aspartic acid backbone with arginine side chains. The aspartic backbone has the potential to replace synthetic polyacrylate, although current production costs are prohibitive. In Chapter 2, a CphA variant from Tatumella morbirosei is characterized, that produces up to 3x more CGP than other known variants, and shows high iCGP specificity in both flask and bioreactor trials. Another CphA variant, this one from Acinetobacter baylyi, underwent rational protein design to create novel mutants. One, G217K, is 34% more productive than the wild type, while G163K produces a CGP with shorter chain lengths. The current structure refined from 4.4Å to 3.5Å. Another exciting application of NRPSs is in healthcare. They can be used to generate novel peptides such as complex antibiotics. A recently discovered iterative polyketide synthase (IPTK), dubbed AlnB, produces an antibiotic called allenomycin. One of the modular subunits, a dehydratase named AlnB_DH, was crystallized to 2.45Å. Several mutations were created in multiple active site residues to help understand the functional mechanism of AlnB_DH. A preliminary holoenzyme AlnB structure at 3.8Å was generated although the large disorganized regions demonstrated an incomplete structure. It was found that chain length is the primary factor in driving dehydratase action within AlnB_DH, which helps lend understanding to this module.
ContributorsSwain, Kyle (Author) / Nannenga, Brent (Thesis advisor) / Nielsen, David (Committee member) / Mills, Jeremy (Committee member) / Seo, Eileen (Committee member) / Acharya, Abhinav (Committee member) / Arizona State University (Publisher)
Created2022
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Description

Esters are important solvents in multiple industries including adhesives, food, and pharmaceuticals. Although esters are biodegradable solvents, the conventional process of producing them is not eco-friendly because they are largely derived from petrochemicals. This has led scientists to consider implementing biological routes in their production process by incorporating heterologous or

Esters are important solvents in multiple industries including adhesives, food, and pharmaceuticals. Although esters are biodegradable solvents, the conventional process of producing them is not eco-friendly because they are largely derived from petrochemicals. This has led scientists to consider implementing biological routes in their production process by incorporating heterologous or improving inherent esterification pathways. However, due to inequality in the biosynthesis of esters and their precursors (organic acid and alcohol), a significant amount of precursors are left unconverted, thereby lowering overall esterification efficiency. Therefore, the primary goal of the current research is to improve the ester titers by incorporating one more step of in vitro esterification with the culture broth, thereby esterifying the unconverted precursors using high efficiency commercial enzymes in the presence of compatible organic solvent. In principle, the medium containing the precursors will be treated with the enzyme in presence of organic solvent, where the precursors will be distributed in both the phases, aqueous and organic, based on their polarity, and the enzymatic esterification will happen at the interface. Hence, as a first step, efforts were made to optimize the reaction conditions, beginning with choosing the most efficient organic solvent and corresponding enzyme candidate. Our results showed that, for production of ethyl acetate through this reactive extraction approach, Novozyme435 exhibited significant esterification with chloroform, with almost 85% conversion efficiency. Further optimizations with phase ratios, pH and incubation time showed that the pH 6.0 (3.1 g/L) was the most optimum where ethyl acetate titer was found to improve 10 times than that at pH 7.0 (0.164 g/L) with the phase ratio of 1:1. The kinetic studies further added that the incubation at 37oC gives the maximum ethyl acetate production within 8h. After initial optimization studies, cell broth from E. coli cells transformed to overproduce an esterase was also tested with the reactive extraction method. It was found that there was a ~7.5X decrease in ethyl acetate production in the cell media versus synthetic samples with the same concentration of reactants. Such a large decrease indicates that enzymatic promiscuity or inhibition currently prevent the cell samples from reaching the same conversion as synthetic studies. To characterize the maximum reaction rate (Vmax) and affinity constants of the substrates to Novozym 435, further kinetic studies were performed with one minute of reaction. The mathematical model employed assumes that enzyme kinetics rather than diffusion was the rate limiting step, that the concentrations of reactants at the interface are equivalent to the initial concentration of reactants, and that neither substrate is an inhibitor. Vmax was found to be 18.5 Mmol min-1g-1 (of catalyst used), and the affinity constants were 0.957 M and 0.00557 M for acetic acid and ethanol respectively. Vmax was similar to literature values with Novozym 435, and the affinity constants indicate a much higher binding efficiency of ethanol in comparison to acetic acid, indicating that a cocktail of esters are likely produced from Novozym 435 in cell broth. Overall, moving away from fossil-fuel dependence is necessary to promote sustainable industry standards, and microbial cell factories combined with reactive extraction, if optimized for industrial applications, can replace harmful environmental procedures. By optimizing the reactive extraction process for ester production, biorefineries could become more competitive and economically feasible for numerous applications.

ContributorsKartchner, Danika (Author) / Varman, Arul Mozhy (Thesis director) / Nielsen, David (Committee member) / Soundappan, Thiagarajan (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / Watts College of Public Service & Community Solut (Contributor)
Created2022-05
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Description
Energy can be harvested from wastewater using microbial fuel cells (MFC). In order to increase power generation, MFCs can be scaled-up. The MFCs are designed with two air cathodes and two anode electrodes. The limiting electrode for power generation is the cathode and in order to maximize power, the cathodes

Energy can be harvested from wastewater using microbial fuel cells (MFC). In order to increase power generation, MFCs can be scaled-up. The MFCs are designed with two air cathodes and two anode electrodes. The limiting electrode for power generation is the cathode and in order to maximize power, the cathodes were made out of a C-N-Fe catalyst and a polytetrafluoroethylene binder which had a higher current production at -3.2 mA/cm2 than previous carbon felt cathodes at -0.15 mA/cm2 at a potential of -0.29 V. Commercial microbial fuel cells from Aquacycl were tested for their power production while operating with simulated blackwater achieved an average of 5.67 mW per cell. The small MFC with the C-N-Fe catalyst and one cathode was able to generate 8.7 mW. Imitating the Aquacycl cells, the new MFC was a scaled-up version of the small MFC where the cathode surface area increased from 81 cm2 to 200 cm2. While the MFC was operating with simulated blackwater, the peak power produced was 14.8 mW, more than the smaller MFC, but only increasing in the scaled-up MFC by 1.7 when the surface area of the cathode increased by 2.46. Further long-term application can be done, as well as operating multiple MFCs in series to generate more power and improve the design.
ContributorsRussell, Andrea (Author) / Torres, Cesar (Thesis advisor) / Garcia Segura, Sergio (Committee member) / Fraser, Matthew (Committee member) / Arizona State University (Publisher)
Created2022
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Description
The current use of non-renewable fossil fuels for industry poses a threat for future generations. Thus, a pivot to renewable sources of energy must be made to secure a sustainable future. One potential option is the utilization of metabolically engineered bacteria to produce value-added chemicals during fermentation. Currently, numerous strains

The current use of non-renewable fossil fuels for industry poses a threat for future generations. Thus, a pivot to renewable sources of energy must be made to secure a sustainable future. One potential option is the utilization of metabolically engineered bacteria to produce value-added chemicals during fermentation. Currently, numerous strains of metabolically engineered Escherichia coli have shown great capacity to specialize in the production of high titers of a desired chemical. These metabolic systems, however, are constrained by the biological limits of E. coli itself. During fermentation, E. coli grows to less than one twentieth of the density that aerobically growing cultures can reach. I hypothesized that this decrease in growth during fermentation is due to cellular stress associated with fermentative growth, likely caused by stress related genes. These genes, including toxin-antitoxin (TA) systems and the rpoS mediated general stress response, may have an impact on fermentative growth constraints. Through transcriptional analysis, I identified that the genes pspC and relE are highly expressed in fermenting strains of both wild type and metabolically engineered E. coli. Fermentation of toxin gene knockouts of E. coli BW25113 revealed their potential impacts on E. coli fermentation. The inactivation of ydcB, lar, relE, hipA, yjfE, chpA, ygiU, ygjN, ygfX, yeeV, yjdO, yjgK and ydcX did not lead to significant changes in cell growth when tested using sealed tubes under microaerobic conditions. In contrast, inactivation of pspC, yafQ, yhaV, yfjG and yoeB increased cell growth after 12 hours while inactivation yncN significantly arrested cell growth in both tube and fermentation tests, thus proving these toxins’ roles in fermentative growth. Moreover, inactivation of rpoS also significantly hindered the ability of E. coli to ferment, suggesting its important role in E. coli fermentation
ContributorsHernandez, Michaella (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022