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- All Subjects: Bioengineering
- Creators: Rittmann, Bruce E.
- Creators: Varman, Arul M
Description
Lignin is a naturally abundant source of aromatic carbon but is largely underutilized inindustry because it is difficult to decompose. Recent research activity has targeted the
development of a biological platform for the conversion of lignin and lignin-derived
feedstock. Corynebacterium glutamicum is a standout candidate for the bacterial
depolymerization and assimilation of lignin because of its performance as an industrial
producer of amino acids, resistance to aromatic compounds in lignin, and low
extracellular protease activity. Under the current study, nine experimental strains of C.
glutamicum were engineered with sequencing-confirmed plasmids to overexpress and
secrete lignin-modifying enzymes with the eventual goal of using lignin as raw feed for
the sustainable production of valuable chemicals. Within the study, laccase and
peroxidase activity were discovered to be decreased in C. glutamicum culture media. For
laccase the decrease reached statistical significance, with an activity of about 10.9 U/L
observed in water but only about 7.56 U/L and 7.42 U/L in fresh and spent BHI media,
respectively, despite the same amounts of enzyme being added. Hypothesized reasons for
this inhibitory effect are discussed here, but further work is needed to identify causative
factors and realize the potential of C. glutamicum for waste biomass valorization.
ContributorsEllis, Dylan Scott (Author) / Varman, Arul M (Thesis advisor) / Lammers, Peter J (Committee member) / Long, Timothy E (Committee member) / Arizona State University (Publisher)
Created2022
Description
Phenolic polymers like polyphenols and polyphenylenes have several industrial applications including electrical insulation, specialty membranes, and packings but are typically synthesized under harsh reaction conditions and require hazardous chemicals like formaldehyde. Hydroxycinnamic acids, such as p-coumaric acid (p-CA), are aromatic derivatives of lignin hydrolysates, an underutilized and promising renewable feedstock for production of phenolics and phenolic polymers. Recently a strain of Corynebacterium glutamicum has been created by the Joint BioEnergy Institute (JBEI) which expresses phenolic acid decarboxylase (PAD), an enzyme which catalyzes the reaction of p-CA to 4-vinylphenol (4-VP). Further, a deletion of the phdA gene prevents assimilation of p-CA, thereby increasing 4-VP yield. 4-VP is a substituted phenol which can be polymerized to poly(4-vinylphenol) (PVP) in the presence of ligninolytic enzymes like laccases or peroxidases. This work explores in situ polymerization of 4-VP to PVP by supplementing ligninolytic enzymes during fermentation. Cultured in the presence of p-CA, the engineered C. glutamicum strain achieved a maximum 4-VP yield of 45.2%, 57.9%, and 34.7% when fed 2, 5, and 10 g/L p-CA, respectively. Low yield can be attributed to photodegradation of 4-VP and accumulation of the native laccase present in C. glutamicum which may form only dimers and trimers. To further investigate carbon utilization in the cell, the engineered strain was plasmid cured thus removing the PAD enzyme and fermentations for 13C pathway analysis was performed. Polymerization experiments were performed and the polymer was characterized using GPC.
ContributorsMcKeown, Haley Nicole (Author) / Varman, Arul M (Thesis advisor) / Nannenga, Brent (Committee member) / Nielsen, David (Committee member) / Arizona State University (Publisher)
Created2024
Description
Metabolic engineering has emerged as a highly effective approach to optimizing industrial fermentation processes by introducing purposeful genetic alterations using recombinant DNA technology. Successful metabolic engineering begins with a careful investigation of cellular function, and based on the outcomes of this analysis, an improved strain is created and then constructed using genetic engineering. By modifying the genetic makeup of cells, can increase the production of important chemicals, biofuels, medications, and agricultural products. The most often used genetic engineering tool is plasmid-based gene editing. In plasmid-based gene editing, the desired gene sequence is flanked by similar genome sequences, which encourages the foreign gene's integration into the genome. The main flaw of plasmid-based editing is the presence of selectable markers in the integrated DNA, which impacts cell stability as well as downstream applications that are critical to industries. Recently, with the growth of science, the new gene-editing technology CRISPR (clustered regularly interspaced short palindromic repeat) has revolutionized the field of gene editing. It has been used to incorporate the foreign genes into the genome of the microbial host without any mark and has more efficiency than the plasmid-based gene editing technique. CRISPR is utilized to achieve markerless integration of genes in genomes of microbes, which promotes cell stability and is also especially beneficial for applications in industries. In this experiment successfully integrated two genes into the genome of C.glutamicum employing markerless integration via homologous recombination, allowing cells to metabolize acetate into acetyl-CoA and improve the conversion of pyruvate into lactate. Further, this strain of C.glutamicum can be utilized as a platform for producing ethyl lactate, a green solvent using a microbial host
ContributorsBrahmankar, Sumant Milind (Author) / Varman, Arul M (Thesis advisor) / Nielsen, David R (Committee member) / Seto, Jong (Committee member) / Arizona State University (Publisher)
Created2024
Description
Petroleum contamination is ubiquitous during extraction, transportation, refining, and storage. Contamination damages the soil’s ecosystem function, reduces its aesthetics, and poses a potential threat to human beings. The overall goals of this dissertation are to advance understanding of the mechanisms behind ozonation of petroleum-contaminated soil and to configure an effective integrated bioremediation + ozonation remedial strategy to remove the overall organic carbon. Using a soil column, I conducted batch ozonation experiments for different soils and at different moisture levels. I measured multiple parameters: e.g., total petroleum hydrocarbons (TPH) and dissolved organic carbon (DOC), to build a full understanding of the data that led to the solid conclusions. I first demonstrated the feasibility of using ozone to attack heavy petroleum hydrocarbons in soil settings. I identified the physical and chemical hurdles (e.g., moisture, mass transfer, pH) needed to be overcome to make the integration of chemical oxidation and biodegradation more efficient and defines the mechanisms behind the experimental observations. Next, I completed a total carbon balance, which revealed that multiple components, including soil organic matter (SOM) and non-TPH petroleum, competed for ozone, although TPH was relatively more reactive. Further experiments showed that poor soil mixing and high soil-moisture content hindered mass transfer of ozone to react with the TPH. Finally, I pursued the theme of optimizing the integration of ozonation and biodegradation through a multi-stage strategy. I conducted multi-stages of ozonation and bioremediation for two benchmark soils with distinctly different oils to test if and how much ozonation enhanced biodegradation and vice versa. With pH and moisture optimized for each step, pre-ozonation versus post-ozonation was assessed for TPH removal and mineralization. Multi-cycle treatment was able to achieve the TPH regulatory standard when biodegradation alone could not. Ozonation did not directly enhance the biodegradation rate of TPH; instead, ozone converted TPH into DOC that was biodegraded and mineralized. The major take-home lesson from my studies is that multi-stage ozonation + biodegradation is a useful remediation tool for petroleum contamination in soil.
ContributorsChen, Tengfei (Author) / Rittmann, Bruce E. (Thesis advisor) / Westerhoff, Paul (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Delgado, Anca G (Committee member) / Arizona State University (Publisher)
Created2018
Description
On average, our society generates ~0.5 ton of municipal solid waste per person annually. Biomass waste can be gasified to generate synthesis gas (syngas), a gas mixture consisting predominantly of CO, CO2, and H2. Syngas, rich in carbon and electrons, can fuel the metabolism of carboxidotrophs, anaerobic microorganisms that metabolize CO (a toxic pollutant) and produce biofuels (H2, ethanol) and commodity chemicals (acetate and other fatty acids). Despite the attempts for commercialization of syngas fermentation by several companies, the metabolic processes involved in CO and syngas metabolism are not well understood. This dissertation aims to contribute to the understanding of CO and syngas fermentation by uncovering key microorganisms and understanding their metabolism. For this, microbiology and molecular biology techniques were combined with analytical chemistry analyses and deep sequencing techniques. First, environments where CO is commonly detected, including the seafloor, volcanic sand, and sewage sludge, were explored to identify potential carboxidotrophs. Since carboxidotrophs from sludge consumed CO 1000 faster than those in nature, mesophilic sludge was used as inoculum to enrich for CO- and syngas- metabolizing microbes. Two carboxidotrophs were isolated from this culture: an acetate/ethanol-producer 99% phylogenetically similar to Acetobacterium wieringae and a novel H2-producer, Pleomorphomonas carboxidotrophicus sp. nov. Comparison of CO and syngas fermentation by the CO-enriched culture and the isolates suggested mixed-culture syngas fermentation as a better alternative to ferment CO-rich gases. Advantages of mixed cultures included complete consumption of H2 and CO2 (along with CO), flexibility under different syngas compositions, functional redundancy (for acetate production) and high ethanol production after providing a continuous supply of electrons. Lastly, dilute ethanol solutions, typical of syngas fermentation processes, were upgraded to medium-chain fatty acids (MCFA), biofuel precursors, through the continuous addition of CO. In these bioreactors, methanogens were inhibited and Peptostreptococcaceae and Lachnospiraceae spp. most likely partnered with carboxidotrophs for MCFA production. These results reveal novel microorganisms capable of effectively consuming an atmospheric pollutant, shed light on the interplay between syngas components, microbial communities, and metabolites produced, and support mixed-culture syngas fermentation for the production of a wide variety of biofuels and commodity chemicals.
ContributorsEsquivel Elizondo, Sofia Victoria (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Rittmann, Bruce E. (Committee member) / Delgado, Anca G. (Committee member) / Torres, Cesar I. (Committee member) / Arizona State University (Publisher)
Created2017
Description
Seeking to address sustainability issues associated with food waste (FW), and fat, oil, and grease (FOG) waste disposal, the City of Mesa commissioned the Biodesign Swette Center for Environmental Biotechnology (BSCEB) at Arizona State University (ASU) to study to the impact of implementing FW/FOG co-digestion at the wastewater treatment plant (WWTP). A key issue for the study was the “souring” of the anaerobic digesters (ADs), which means that the microorganism responsible for organic degradation were deactivated, causing failure of the AD. Several bench-scale reactors soured after the introduction of the FW/FOG feed streams. By comparing measurements from stable with measurements from the souring reactors, I identified two different circumstances responsible for souring events. One set of reactors soured rapidly after the introduction of FW/FOG due to the digester’s hydraulic retention times (HRT) becoming too short for stable operation. A second set of reactors soured after a long period of stability due to steady accumulation of fatty acids (FAs) that depleted bicarbonate alkalinity. FA accumulation was caused by the incomplete hydrolysis/fermentation of feedstock protein, leading to insufficient release of ammonium (NH4+). In contrast, carbohydrates were more rapidly hydrolyzed and fermented to FAs.
The most important contribution of my research is that I identified several leading indicators of souring. In all cases of souring, the accumulation of soluble chemical oxygen demand (SCOD) was an early and easily quantified indicator. A shift in effluent FA concentrations from shorter to longer species also portended souring. A reduction in the yield of methane (CH4) per mass of volatile suspended solids removed (VSSR) also identified souring conditions, but its variability prevented the methane yield from providing advanced warning to allow intervention. For the rapidly soured reactors, reduced bicarbonate alkalinity was the most useful warning sign, and an increasing ratio of SCOD to bicarbonate alkalinity was the clearest sign of souring. Because I buffered the slow-souring reactors with calcium carbonate (CaCO3), I could not rely on bicarbonate alkalinity as an indicator, which put a premium on SCOD as the early warning. I implemented two buffering regimes and demonstrated that early and consistent buffering could lead to reactor recovery.
The most important contribution of my research is that I identified several leading indicators of souring. In all cases of souring, the accumulation of soluble chemical oxygen demand (SCOD) was an early and easily quantified indicator. A shift in effluent FA concentrations from shorter to longer species also portended souring. A reduction in the yield of methane (CH4) per mass of volatile suspended solids removed (VSSR) also identified souring conditions, but its variability prevented the methane yield from providing advanced warning to allow intervention. For the rapidly soured reactors, reduced bicarbonate alkalinity was the most useful warning sign, and an increasing ratio of SCOD to bicarbonate alkalinity was the clearest sign of souring. Because I buffered the slow-souring reactors with calcium carbonate (CaCO3), I could not rely on bicarbonate alkalinity as an indicator, which put a premium on SCOD as the early warning. I implemented two buffering regimes and demonstrated that early and consistent buffering could lead to reactor recovery.
ContributorsKupferer III, Rick Anthony (Author) / Rittmann, Bruce E. (Thesis advisor) / Young, Michelle N (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2020
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
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