Matching Items (16)
Filtering by
- Creators: Arizona State University
- Creators: Nielsen, David
Description
With the aid of metabolic pathways engineering, microbes are finding increased use as biocatalysts to convert renewable biomass resources into fine chemicals, pharmaceuticals and other valuable compounds. These alternative, bio-based production routes offer distinct advantages over traditional synthesis methods, including lower energy requirements, rendering them as more "green" and "eco-friendly". Escherichia coli has recently been engineered to produce the aromatic chemicals (S)-styrene oxide and phenol directly from renewable glucose. Several factors, however, limit the viability of this approach, including low titers caused by product inhibition and/or low metabolic flux through the engineered pathways. This thesis focuses on addressing these concerns using magnetic mesoporous carbon powders as adsorbents for continuous, in-situ product removal as a means to alleviate such limitations. Using process engineering as a means to troubleshoot metabolic pathways by continuously removing products, increased yields are achieved from both pathways. By performing case studies in product toxicity and reaction equilibrium it was concluded that each step of a metabolic pathway can be optimized by the strategic use of in-situ adsorption as a process engineering tool.
ContributorsVasudevan, Anirudh (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2014
Description
Synthetic biology is constantly evolving as new ideas are incorporated into this increasingly flexible field. It incorporates the engineering of life with standard genetic parts and methods; new organisms with new genomes; expansion of life to include new components, capabilities, and chemistries; and even completely synthetic organisms that mimic life while being composed of non-living matter. We have introduced a new paradigm of synthetic biology that melds the methods of in vitro evolution with the goals and philosophy of synthetic biology. The Family B proteins represent the first de novo evolved natively folded proteins to be developed with increasingly powerful tools of molecular evolution. These proteins are folded and functional, composed of the 20 canonical amino acids, and in many ways resemble natural proteins. However, their evolutionary history is quite different from natural proteins, as it did not involve a cellular environment. In this study, we examine the properties of DX, one of the Family B proteins that have been evolutionarily optimized for folding stability. Described in chapter 2 is an investigation into the primitive catalytic properties of DX, which seems to have evolved a serendipitous ATPase activity in addition to its selected ATP binding activity. In chapters 3 and 4 we express the DX gene in E. coli cells and observe massive changes in cell morphology, biochemistry, and life cycle. Exposure to DX activates several defense systems in E. coli, including filamentation, cytoplasmic segregation, and reversion to a viable but non-culturable state. We examined these phenotypes in detail and present a model that accounts for how DX causes such a rearrangement of the cell.
ContributorsStomel, Joshua (Author) / Chaput, John C (Thesis advisor) / Korch, Shaleen (Committee member) / Roberson, Robert (Committee member) / Ghirlanda, Gionvanna (Committee member) / Arizona State University (Publisher)
Created2011
Description
Depletion of fossil fuel resources has led to the investigation of alternate feedstocks for and methods of chemical synthesis, in particular the use of E. coli biocatalysts to produce fine commodity chemicals from renewable glucose sources. Production of phenol, 2-phenylethanol, and styrene was investigated, in particular the limitation in yield and accumulation that results from high product toxicity. This paper examines two methods of product toxicity mitigation: the use of efflux pumps and the separation of pathways which produce less toxic intermediates. A library of 43 efflux pumps from various organisms were screened for their potential to confer resistance to phenol, 2-phenylethanol, and styrene on an E. coli host. A pump sourced from P. putida was found to allow for increased host growth in the presence of styrene as compared to a cell with no efflux pump. The separation of styrene producing pathway was also investigated. Cells capable of performing the first and latter halves of the synthesis were allowed to grow separately and later combined in order to capitalize on the relatively lower toxicity of the intermediate, trans-cinnamate. The styrene production and yield from this separated set of cultures was compared to that resulting from the growth of cells containing the full set of styrene synthesis genes. Results from this experiment were inconclusive.
ContributorsLallmamode, Noor Atiya Jabeen (Author) / Nielsen, David (Thesis director) / Cadillo-Quiroz, Hinsby (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / School of Life Sciences (Contributor)
Created2015-05
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. 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
Description
The goals of the styrene oxide adsorption experiments were to develop reliable isotherms of styrene oxide onto Dowex Optipore L-493 resin and onto mesoporous carbon adsorbents, in addition to determining the ideal conditions for styrene oxide production from E. coli. Adsorption is an effective means of separation used in industry to separate compounds, often organics from air and water. Styrene oxide adsorption runs without E. coli were conducted at concentrations ranging from 0.15 to 3.00 g/L with resin masses ranging from 0.1 to 0.5 g of Dowex Optipore L-493 and 0.5 to 0.75 g of mesoporous carbon adsorbent. Runs were conducted on a shake plate operating at 80 rpm for 24 hours at ambient temperature. Isotherms were developed from the results and then adsorption experiments with E. coli and L-493 were performed. Runs were conducted at glucose concentrations ranging from 20-40 g/L and resin masses of 0.100 g to 0.800 g. Samples were incubated for 72 hours and styrene oxide production was measured using an HPLC device. Specific loading values reached up to 0.356 g/g for runs without E. coli and nearly 0.003 g of styrene oxide was adsorbed by L-493 during runs with E. coli. Styrene oxide production was most effective at low resin masses and medium glucose concentrations when produced by E. coli.
ContributorsHsu, Joshua (Co-author) / Oremland, Zachary (Co-author) / Nielsen, David (Thesis director) / Staggs, Kyle (Committee member) / Barrett, The Honors College (Contributor) / Chemical Engineering Program (Contributor) / School of Sustainability (Contributor)
Created2014-05
Description
The inability of a single strain of bacteria to simultaneously and completely consume multiple sugars, such as glucose and xylose, hinder industrial microbial processes for ethanol and lactate production. To overcome this limitation, I am engineering an E. coli co-culture system consisting of two ‘specialists'. One has the ability to only consume xylose and the other only glucose. This allows for co-utilization of lignocellulose-derived sugars so both sugars are completely consumed, residence time is reduced and lactate and ethanol titers are maximized.
ContributorsAyla, Zeynep Ece (Author) / Nielsen, David (Thesis director) / Flores, Andrew (Committee member) / Chemical Engineering Program (Contributor) / School of Sustainability (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
Escherichia coli is a bacterium that is used widely in metabolic engineering due to its ability to grow at a fast rate and to be cultured easily. E. coli can be engineered to produce many valuable chemicals, including biofuels and L-Phenylalanine—a precursor to many pharmaceuticals. Significant cell growth occurs in parallel to the biosynthesis of the desired biofuel or biochemical product, and limits product concentrations and yields. Stopping cell growth can improve chemical production since more resources will go toward chemical production than toward biomass. The goal of the project is to test different methods of controlling microbial uptake of nutrients, specifically phosphate, to dynamically limit cell growth and improve biochemical production of E. coli, and the research has the potential to promote public health, sustainability, and environment. This can be achieved by targeting phosphate transporter genes using CRISPRi and CRISPR, and they will limit the uptake of phosphate by targeting the phosphate transporter genes in DNA, which will stop transcriptions of the genes. In the experiment, NST74∆crr∆pykAF, a L-Phe overproducer, was used as the base strain, and the pitA phosphate transporter gene was targeted in the CRISPRi and CRISPR systems with the strain with other phosphate transporters knocked out. The tested CRISPRi and CRISPR mechanisms did not stop cell growth or improved L-Phe production. Further research will be conducted to determine the problem of the system. In addition, the CRISPRi and CRISPR systems that target multiple phosphate transporter genes will be tested in the future as well as the other method of stopping transcriptions of the phosphate transporter genes, which is called a tunable toggle switch mechanism.
ContributorsPark, Min Su (Author) / Nielsen, David (Thesis director) / Machas, Michael (Committee member) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The need for rapid, specific and sensitive assays that provide a detection of bacterial indicators are important for monitoring water quality. Rapid detection using biosensor is a novel approach for microbiological testing applications. Besides, validation of rapid methods is an obstacle in adoption of such new bio-sensing technologies. In this study, the strategy developed is based on using the compound 4-methylumbelliferyl glucuronide (MUG), which is hydrolyzed rapidly by the action of E. coli β-D-glucuronidase (GUD) enzyme to yield a fluorogenic product that can be quantified and directly related to the number of E. coli cells present in water samples. The detection time required for the biosensor response ranged from 30 to 120 minutes, depending on the number of bacteria. The specificity of the MUG based biosensor platform assay for the detection of E. coli was examined by pure cultures of non-target bacterial genera and also non-target substrates. GUD activity was found to be specific for E. coli and no such enzymatic activity was detected in other species. Moreover, the sensitivity of rapid enzymatic assays was investigated and repeatedly determined to be less than 10 E. coli cells per reaction vial concentrated from 100 mL of water samples. The applicability of the method was tested by performing fluorescence assays under pure and mixed bacterial flora in environmental samples. In addition, the procedural QA/QC for routine monitoring of drinking water samples have been validated by comparing the performance of the biosensor platform for the detection of E. coli and culture-based standard techniques such as Membrane Filtration (MF). The results of this study indicated that the fluorescence signals generated in samples using specific substrate molecules can be utilized to develop a bio-sensing platform for the detection of E. coli in drinking water. The procedural QA/QC of the biosensor will provide both industry and regulatory authorities a useful tool for near real-time monitoring of E. coli in drinking water samples. Furthermore, this system can be applied independently or in conjunction with other methods as a part of an array of biochemical assays in order to reliably detect E. coli in water.
ContributorsHesari, Nikou (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2015
Description
Advanced oxidation processes (AOP’s) are water/wastewater treatment processes simultaneously providing disinfection and potential oxidation of contaminants that may cause long-term adverse health effects in humans. One AOP involves injecting peracetic acid (PAA) upstream of an ultraviolet (UV) irradiation reactor. Two studies were conducted, one in pilot-scale field conditions and another under laboratory conditions. A pilot-scale NeoTech UV reactor (rated for 375 GPM) was used in the pilot study, where a smaller version of this unit was used in the laboratory study (20 to 35 GPM). The pilot study analyzed coliform disinfection and also monitored water quality parameters including UV transmittance (UVT), pH and chlorine residual. Pilot study UV experiments indicate the unit is effectively treating flow streams (>6 logs total coliforms) twice the 95% UVT unit capacity (750 GPM or 17 mJ/cm2 UV Dose). The results were inconclusive on PAA/UV inactivation due to high data variability and field operation conditions creating low inlet concentrations.Escherichia coli (E. coli) bacteria and the enterobacteria phage P22—a surrogate for enteric viruses—were analyzed. UV inactivated >7.9 and 4 logs of E. coli and P22 respectively at a 16.8 mJ/cm2 UV dose in test water containing a significant organics concentration. When PAA doses of 0.25 and 0.5 mg/L were injected upstream of UV at approximately the same UV Dose, the average E.coli log inactivation increased to >8.9 and >9 logs respectively, but P22 inactivation decreased to 2.9 and 3.0 logs, respectively. A bench-scale study with PAA was also conducted for 5, 10 and 30 minutes of contact time, where 0.25 and 0.5 mg/L had <1 log inactivation of E. coli and P22 after 30 minutes of contact time. In addition, degradation of the chemical N-Nitrosodimethylamine (NDMA) in tap water was analyzed, where UV degraded NDMA by 48 to 97% for 4 and 0.5 GPM flowrates, respectively. Adding 0.5 mg/L PAA upstream of UV did not significantly improve NDMA degradation.
The results under laboratory conditions indicate that PAA/UV have synergy in the inactivation of bacteria, but decrease virus inactivation. In addition, the pilot study demonstrates the applicability of the technology for full scale operation.
The results under laboratory conditions indicate that PAA/UV have synergy in the inactivation of bacteria, but decrease virus inactivation. In addition, the pilot study demonstrates the applicability of the technology for full scale operation.
ContributorsCooper, Samantha (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2017
Description
This study was designed to provide insight into microbial transport kinetics which might be applied to bioremediation technology development and prevention of groundwater susceptibility to pathogen contamination. Several pilot-scale experiments were conducted in a saturated, 2 dimensional, packed porous media tank to investigate the transport of Escherichia coli bacteria, P22 bacteriophage, and a visual tracer and draw comparisons and/or conclusions. A constructed tank was packed with an approximate 3,700 cubic inches (in3) of a fine grained, homogeneous, chemically inert sand which allowed for a controlled system. Sampling ports were located at 5, 15, 25, and 25 vertical inches from the base of the 39 inch saturated zone and were used to assess the transport of the selected microorganisms. Approximately 105 cells of E. coli or P22 were injected into the tank and allowed to move through the media at approximately 10.02 inches per day. Samples were collected intermittently after injection based off of an estimated sampling schedule established from the visual tracer.
The results suggest that bacteriophages pass through soil faster and with greater recovery than bacteria. P22 in the tank reservoir experienced approximately 1 log reduction after 36 hours. After 85 hours, P22 was still detected in the reservoir after experiencing a 2 log reduction from the start of the experiment. E. coli either did not reach the outlet or died before sampling, while P22 was able to be recovered. Bacterial breakthrough curves were produced for the microbial indicators and illustrate the peak concentrations found for each sampling port. For E. coli, concentrations at the 5 inch port peaked at a maximum of 5170 CFU/mL, and eventually at the 25 inch port at a maximum of 90 CFU/mL. It is presumed that E. coli might have experienced significant filtration, straining and attachment, while P22 might have experienced little adsorption and instead was transported rapidly in long distances and was able to survive for the duration of the experiment.
The results suggest that bacteriophages pass through soil faster and with greater recovery than bacteria. P22 in the tank reservoir experienced approximately 1 log reduction after 36 hours. After 85 hours, P22 was still detected in the reservoir after experiencing a 2 log reduction from the start of the experiment. E. coli either did not reach the outlet or died before sampling, while P22 was able to be recovered. Bacterial breakthrough curves were produced for the microbial indicators and illustrate the peak concentrations found for each sampling port. For E. coli, concentrations at the 5 inch port peaked at a maximum of 5170 CFU/mL, and eventually at the 25 inch port at a maximum of 90 CFU/mL. It is presumed that E. coli might have experienced significant filtration, straining and attachment, while P22 might have experienced little adsorption and instead was transported rapidly in long distances and was able to survive for the duration of the experiment.
ContributorsAcosta, Jazlyn Cauren (Author) / Abbaszadegan, Morteza (Thesis advisor) / Dahlen, Paul (Committee member) / Fox, Peter (Committee member) / Arizona State University (Publisher)
Created2017