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- All Subjects: chemical engineering
- Creators: Nielsen, David R
Description
The aims of this project are to demonstrate the design and implementation of separations modalities for 1) in situ product recovery and 2) upstream pretreatment of toxic feedstocks. Many value-added bioproducts such as alcohols (ethanol and butanol) developed for the transportation sector are known to be integral to a sustainable future. Likewise, bioproduced aromatic building blocks for sustainable manufacturing such as phenol will be equally important. The production of these compounds is often limited by product toxicity at 2- 20 g/L, whereas it may desirable to produce 20-200 g/L for economically feasible scale up. While low-cost feedstocks are desirable for economical production, they contain highly cytotoxic value-added byproducts such as furfural. It is therefore desirable to design facile detoxification methods for lignocellulose-derived feedstocks to isolate and recover furfural preceding ethanol fermentation by Escherichia coli. Correspondingly it is desirable to design efficient facile in situ recovery modalities for bioalcohols and phenolic bioproducts. Accordingly, in-situ removal modalities were designed for simultaneous acetone, butanol, and ethanol recovery. Additionally, a furfural removal modality from lignocellulosic hydrolysates was designed for upstream pretreatment. Solid-liquid adsorption was found to serve well each of the recovery modalities characterized here. More hydrophobic compounds such as butanol and furfural are readily recovered from aqueous solutions via adsorption. The primary operational drawback to adsorption is adsorbent recovery and subsequent desorption of the product. Novel magnetically separable mesoporous carbon powders (MMCPs) were characterized and found to be rapidly separable from solutions at 91% recovery by mass. Thermal desorption of value added products was found efficient for recovery of butanol and furfural. Fufural was desorbed from the MMCPs up to 57% by mass with repeated adsorption/thermal desorption cycles. Butanol was recovered from MMCPs up to an average 93% by mass via thermal desorption. As another valuable renewable fermentation product, phenol was also collected via in-situ adsorption onto Dowex Optipore L-493 resin. Phenol recovery from the resins was efficiently accomplished with tert-butyl methyl ether up to 77% after 3 washes.
ContributorsStaggs, Kyle William (Author) / Nielsen, David R (Thesis advisor) / Lin, Jerry S (Committee member) / Torres, César I (Committee member) / Lind, Mary Laura (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2017
Description
The engineering of microbial cell factories capable of synthesizing industrially relevant chemical building blocks is an attractive alternative to conventional petrochemical-based production methods. This work focuses on the novel and enhanced biosynthesis of phenol, catechol, and muconic acid (MA). Although the complete biosynthesis from glucose has been previously demonstrated for all three compounds, established production routes suffer from notable inherent limitations. Here, multiple pathways to the same three products were engineered, each incorporating unique enzyme chemistries and/or stemming from different endogenous precursors. In the case of phenol, two novel pathways were constructed and comparatively evaluated, with titers reaching as high as 377 ± 14 mg/L at a glucose yield of 35.7 ± 0.8 mg/g. In the case of catechol, three novel pathways were engineered with titers reaching 100 ± 2 mg/L. Finally, in the case of MA, four novel pathways were engineered with maximal titers reaching 819 ± 44 mg/L at a glucose yield of 40.9 ± 2.2 mg/g. Furthermore, the unique flexibility with respect to engineering multiple pathways to the same product arises in part because these compounds are common intermediates in aromatic degradation pathways. Expanding on the novel pathway engineering efforts, a synthetic ‘metabolic funnel’ was subsequently constructed for phenol and MA, wherein multiple pathways were expressed in parallel to maximize carbon flux toward the final product. Using this novel ‘funneling’ strategy, maximal phenol and MA titers exceeding 0.5 and 3 g/L, respectively, were achieved, representing the highest achievable production metrics products reported to date.
ContributorsThompson, Brian (Author) / Nielsen, David R (Thesis advisor) / Nannenga, Brent (Committee member) / Green, Matthew (Committee member) / Wang, Xuan (Committee member) / Moon, Tae Seok (Committee member) / Arizona State University (Publisher)
Created2017
Description
The project aims at utilization of hydrothermal liquefaction (HTL) byproducts like biochar to grow microalgae. HTL is a promising method to convert wet algal biomasses into biofuels. The initial microalgae liquefaction at a temperature of 300 °C for 30 minute, converted 31.22 % of the Galdieria sulphuraria and 41.00 % of the Kirchneriella cornutum into biocrude. Upon changing the reactor from a 100 ml to a 250 ml reactor, the yield in biocrude increased to 31.48 % for G. sulphuraria and dropped to 38.05 % for K. cornutum. Further, energy recoveries based on calorific values of HTL products were seen to drop by about 5 % of the 100 ml calculated values in the larger reactor.
Biochar from HTL of G. sulphuraria at 300 °C showed 15.98 and 5.27 % of phosphorous and nitrogen, respectively. HTL products from the biomass were analyzed for major elements through ICP-OES and CHNS/O. N and P are macronutrients that can be utilized in growing microalgae. This could reduce the operational demands in growing algae like, phosphorous mined to meet annual national demand for aviation fuel. Acidic leaching of these elements as phosphates and ammoniacal nitrogen was studied. Improved leaching of 49.49 % phosphorous and 95.71 % nitrogen was observed at 40 °C and pH 2.5 over a period of 7 days into the growth media. These conditions being ideal for growth of G. sulphuraria, leaching can be done in-situ to reduce overhead cost.
Growth potential of G. sulphuraria in leached media was compared to a standard cyanidium media produced from inorganic chemicals. Initial inhibition studies were done in the leached media at 40 °C and 2-3 vol. % CO2 to observe a positive growth rate of 0.273 g L-1 day-1. Further, growth was compared to standard media with similar composition in a 96 well plate 50 μL microplate assay for 5 days. The growth rates in both media were comparable. Additionally, growth was confirmed in a 240 times larger tubular reactor in a Tissue Culture Roller drum apparatus. A better growth was observed in the leached cyanidium media as compared to the standard variant.
Biochar from HTL of G. sulphuraria at 300 °C showed 15.98 and 5.27 % of phosphorous and nitrogen, respectively. HTL products from the biomass were analyzed for major elements through ICP-OES and CHNS/O. N and P are macronutrients that can be utilized in growing microalgae. This could reduce the operational demands in growing algae like, phosphorous mined to meet annual national demand for aviation fuel. Acidic leaching of these elements as phosphates and ammoniacal nitrogen was studied. Improved leaching of 49.49 % phosphorous and 95.71 % nitrogen was observed at 40 °C and pH 2.5 over a period of 7 days into the growth media. These conditions being ideal for growth of G. sulphuraria, leaching can be done in-situ to reduce overhead cost.
Growth potential of G. sulphuraria in leached media was compared to a standard cyanidium media produced from inorganic chemicals. Initial inhibition studies were done in the leached media at 40 °C and 2-3 vol. % CO2 to observe a positive growth rate of 0.273 g L-1 day-1. Further, growth was compared to standard media with similar composition in a 96 well plate 50 μL microplate assay for 5 days. The growth rates in both media were comparable. Additionally, growth was confirmed in a 240 times larger tubular reactor in a Tissue Culture Roller drum apparatus. A better growth was observed in the leached cyanidium media as compared to the standard variant.
ContributorsMathew, Melvin (Author) / Deng, Shuguang (Thesis advisor) / Lammers, Peter J. (Committee member) / Nielsen, David R (Committee member) / Arizona State University (Publisher)
Created2017
Description
The release of organophosphorus compounds (OPs) and subsequent exposure to these compounds is of concern to humans and the environment. The goal of this work was to control the concentrations of gaseous OPs through interaction with sorbent oxides. Experimental and computational methods were employed to assess the interactions of dimethyl phosphite (DMHP), dimethyl methylphosphonate (DMMP), dimethyl ethylphosphonate (DMEP), diethyl ethylphosphonate (DEEP), and triethyl phosphate (TEP) with amorphous silica (a-silica), ã-alumina, and monoclinic zirconia (m-zirconia) for applications in air pollution control. Interactions of the selected OPs with a-silica were chosen as a baseline to determine the applicability of the computational predictions. Based on the a-silica results, computational methods were deemed valid for predicting the trends among materials with comparable interactions (e.g. -OH functionality of a-silica interacting with the phosphonyl O atoms of the OPs). Computational evaluations of the interactions with the OPs were extended to the oxide material, m-zirconia, and compared with the results for ã-alumina. It was hypothesized that m-zirconia had the potential to provide for the effective sorption of OPs in a manner superior to that of the a-silica and the ã-alumina surfaces due to the surface charges of the zirconium Lewis acid sites when coordinated in the oxidized form. Based on the computational study, the predicted heats of adsorption for the selected OPs onto m-zirconia were more favorable than those that were predicted for ã-alumina and a-silica. Experimental studies were carried out to confirm these computational results. M-zirconia nanoparticles were synthesized to determine if the materials could be utilized for the adsorption of the selected OPs. M-zirconia was shown to adsorb the OPs, and the heats of adsorption were stronger than those determined for commercial samples of a-silica. However, water interfered with the adsorption of the OPs onto m-zirconia, thus leading to heats of adsorption that were much weaker than those predicted computationally. Nevertheless, this work provides a first investigation of m-zirconia as a viable sorbent material for the ambient control of the selected gaseous OPs. Additionally, this work represents the first comparative study between computational predictions and experimental determination of thermodynamic properties for the interactions of the selected OPs and oxide surfaces.
ContributorsSiu, Eulalia Yuen-Yi (Author) / Andino, Jean M (Thesis advisor) / Forzani, Erica S (Committee member) / Hristovski, Kiril (Committee member) / Nielsen, David R (Committee member) / Pfeffer, Robert (Committee member) / Arizona State University (Publisher)
Created2011
Description
Amino acids and related targets are typically produced by well-characterized heterotrophs including Corynebacterium glutamicum and Escherichia coli. Recent efforts have sought to supplant these sugar-intensive processes through the metabolic engineering of cyanobacteria, which instead can directly utilize atmospheric carbon dioxide (CO2) and sunlight. One of the most promising among recently discovered photoautotrophic strains is Synechococcus elongatus UTEX 2973 (hereafter UTEX 2973), which has been reported to have doubling times as low as 1.5 hours. While encouraging, there are still major challenges preventing the widespread industrial acceptance of engineered cyanobacteria, chief among them is the scarcity of genetic tools and parts with which to engineer production strains. Here, UTEX 2973 was engineered to overproduce L-lysine through the heterologous expression of feedback-resistant copies of aspartokinase lysC and the L-lysine exporter ybjE from Escherichia coli, as aided by the characterization of novel combinations of genetic parts and expression sites. At maximum, using a plasmid-based expression system, a L-lysine titer of 556 ± 62.3 mg/L was attained after 120 hours, surpassing a prior report of photoautotrophic L-lysine bioproduction. Modular extension of the pathway then led to the novel photosynthetic production of the corresponding diamine cadaverine (55.3 ± 6.7 mg/L by 96 hours) and dicarboxylate glutarate (67.5 ± 2.2 mg/L by 96 hours). Lastly, mass transfer experiments were carried out to determine if the solubility of CO2 in and its rate of mass transfer to BG-11 media could be improved by supplementing it with various amines, including cadaverine. Ultimately, however, cyanobacteria grown in the presence of all tested amines was worse than in BG-11 alone, demonstrating the need for additional tolerance engineering to successfully implement this strategy.
ContributorsDookeran, Zachary Anthony (Author) / Nielsen, David R (Thesis advisor) / Wang, Xuan (Committee member) / Nannenga, Brent L (Committee member) / Varman, Arul M (Committee member) / Peebles, Christie AM (Committee member) / Arizona State University (Publisher)
Created2022
Description
Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation.
ContributorsHerschel, Daniel Jordan (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016
Description
Aromatic compounds have traditionally been generated via petroleum feedstocks and have wide ranging applications in a variety of fields such as cosmetics, food, plastics, and pharmaceuticals. Substantial improvements have been made to sustainably produce many aromatic chemicals from renewable sources utilizing microbes as bio-factories. By assembling and optimizing native and non-native pathways to produce natural and non-natural bioproducts, the diversity of biochemical aromatics which can be produced is constantly being improved upon. One such compound, 2-Phenylethanol (2PE), is a key molecule used in the fragrance and food industries, as well as a potential biofuel. Here, a novel, non-natural pathway was engineered in Escherichia coli and subsequently evaluated. Following strain and bioprocess optimization, accumulation of inhibitory acetate byproduct was reduced and 2PE titers approached 2 g/L – a ~2-fold increase over previously implemented pathways in E. coli. Furthermore, a recently developed mechanism to
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.
allow E. coli to consume xylose and glucose, two ubiquitous and industrially relevant microbial feedstocks, simultaneously was implemented and systematically evaluated for its effects on L-phenylalanine (Phe; a precursor to many microbially-derived aromatics such as 2PE) production. Ultimately, by incorporating this mutation into a Phe overproducing strain of E. coli, improvements in overall Phe titers, yields and sugar consumption in glucose-xylose mixed feeds could be obtained. While upstream efforts to improve precursor availability are necessary to ultimately reach economically-viable production, the effect of end-product toxicity on production metrics for many aromatics is severe. By utilizing a transcriptional profiling technique (i.e., RNA sequencing), key insights into the mechanisms behind styrene-induced toxicity in E. coli and the cellular response systems that are activated to maintain cell viability were obtained. By investigating variances in the transcriptional response between styrene-producing cells and cells where styrene was added exogenously, better understanding on how mechanisms such as the phage shock, heat-shock and membrane-altering responses react in different scenarios. Ultimately, these efforts to diversify the collection of microbially-produced aromatics, improve intracellular precursor pools and further the understanding of cellular response to toxic aromatic compounds, give insight into methods for improved future metabolic engineering endeavors.
ContributorsMachas, Michael (Author) / Nielsen, David R (Thesis advisor) / Haynes, Karmella (Committee member) / Wang, Xuan (Committee member) / Nannenga, Brent (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2019
Description
Metabolic engineering of bacteria has become a viable technique as a sustainable and efficient method for the production of biochemicals. Two main goals were explored: investigating styrene tolerance genes in E. coli and engineering cyanobacteria for the high yield production of L-serine. In the first study, genes that were shown to be highly differentially expressed in E. coli upon styrene exposure were further investigated by testing the effects of their deletion and overexpression on styrene tolerance and growth. It was found that plsX, a gene responsible for the phospholipid formation in membranes, had the most promising results when overexpressed at 10 µM IPTG, with a relative OD600 of 706 ± 117% at 175 mg/L styrene when compared to the control plasmid at the same concentration. This gene is likely to be effective target when engineering styrene- and other aromatic-producing strains, increasing titers by reducing their cytotoxicity.In the second study, the goal is to engineer the cyanobacterium Synechococcus sp. PCC 7002 for the overproduction of L-serine. As a robust, photosynthetic bacteria, it has potential for being used in such-rich states to capture CO2 and produce industrially relevant products. In order to increase L-serine titers, a key degradation gene, ilvA, must be removed. While ilvA is responsible for degrading L-serine into pyruvate, it is also responsible for initiating the only known pathway for the production of isoleucine. Herein, we constructed a plasmid containing the native A0730 gene in order to investigate its potential to restore isoleucine production. If functional, a Synechococcus sp. PCC 7002 ΔilvA strain can then be engineered with minimal effects on growth and an expected increase in L-serine accumulation.
ContributorsAbed, Omar (Author) / Nielsen, David R (Thesis advisor) / Varman, Arul M (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2021
Description
Bioconversion of lignocellulosic sugars is often suboptimal due to global regulatory mechanisms such as carbon catabolite repression and incomplete/inefficient metabolic pathways. While conventional bioprocessing strategies for metabolic engineering have predominantly focused on a single engineered strain, the alternative development of synthetic microbial communities facilitates the execution of complex metabolic tasks by exploiting unique community features (i.e., modularity, division of labor, and facile tunability). In this dissertation, these features are leveraged to develop a suite of generalizable strategies and transformative technologies for engineering Escherichia coli coculture systems to more efficiently utilize lignocellulosic sugar mixtures. This was achieved by rationally pairing and systematically engineering catabolically-orthogonal Escherichia coli sugar specialists. Coculture systems were systematically engineered, as derived from either wild-type Escherichia coli W, ethanologenic LY180, lactogenic TG114 or succinogenic KJ122. Net catabolic activities were then readily balanced by simple tuning of the inoculum ratio between sugar specialists, ultimately enabling improved co-utilization (98% of 100 g L-1 total sugars) of glucose-xylose mixtures (2:1 by mass) under simple batch fermentation conditions. We next extended this strategy to a coculture-coproduction system capable of capturing and fixing CO2 evolved during biofuel production through inter-strain metabolic cooperation. Holistically, this work contributes to an improved understanding of the dynamic behavior of synthetic microbial consortia as enhanced bioproduction platforms and carbon conservation strategy for renewable fuels and chemicals from non-food carbohydrates
ContributorsFlores, Andrew David (Author) / Nielsen, David R (Thesis advisor) / Wang, Xuan (Thesis advisor) / Varman, Arul M (Committee member) / Nannenga, Brent (Committee member) / Wheeldon, Ian (Committee member) / Arizona State University (Publisher)
Created2021
Description
Cyanobacteria contribute to more than a quarter of the primary carbon fixation worldwide. They have evolved a CO2 concentrating mechanism (CCM) to enhance photosynthesis because inorganic carbon species are limited in the aqueous environment. Bicarbonate transporters SbtA and BicA are active components of CCM, and the determination of their structures is important to investigate the bicarbonate transport mechanisms. E. coli was selected as the expression host for these bicarbonate transporters, and optimization of expression and protein purification conditions was performed. Single particle electron cryomicroscopy (cryo-EM) or protein crystallography was carried out for each transporter. In this work, SbtA, BicA and SbtB, a regulator protein of SbtA, were heterologously expressed in E. coli and purified for structural studies. SbtB was highly expressed and two different crystal structures of SbtB were resolved at 2.01 Å and 1.8 Å, showing a trimer and dimer in the asymmetric unit, respectively. The yields of SbtA and BicA after purification reached 0.1 ± 0.04 and 6.5 ± 1.0 mg per liter culture, respectively. Single particle analysis showed a trimeric conformation of purified SbtA and promising interaction between SbtA and SbtB, where the bound SbtB was also possibly trimeric. For some crystallization experiments of these transporters, lipidic cubic phase (LCP) was used. In the case of LCP, often times the crystals grown are generally too tiny to withstand radiation damage from the X-ray beam during an X-ray diffraction experiment. As an alternative approach for this research, the microcrystal electron diffraction (MicroED) method was applied to the LCP-laden crystals because it is a powerful cryo-EM method for high-resolution structure determination from protein microcrystals. The new technique is termed as LCP-MicroED, however, prior to applying LCP-MicroED to the bicarbonate transporters, methods needed to be developed for LCP-MicroED. Therefore the model protein Proteinase K was used and its structure was determined to 2.0 Å by MicroED. Additionally, electron diffraction data from cholesterol and human A2A adenosine receptor crystals were collected at 1.0 Å and 4.5 Å using LCP-MicroED, respectively. Other applications of MicroED to different samples are also discussed.
ContributorsBu, Guanhong (Author) / Nannenga, Brent L (Thesis advisor) / Chiu, Po-Lin (Committee member) / Mills, Jeremy H (Committee member) / Nielsen, David R (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2021