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Description
Large-scale cultivation of photosynthetic microorganisms for the production of biodiesel and other valuable commodities must be made more efficient. Recycling the water and nutrients acquired from biomass harvesting promotes a more sustainable and economically viable enterprise. This study reports on growing the cyanobacterium Synechocystis sp. PCC 6803 using

Large-scale cultivation of photosynthetic microorganisms for the production of biodiesel and other valuable commodities must be made more efficient. Recycling the water and nutrients acquired from biomass harvesting promotes a more sustainable and economically viable enterprise. This study reports on growing the cyanobacterium Synechocystis sp. PCC 6803 using permeate obtained from concentrating the biomass by cross-flow membrane filtration. I used a kinetic model based on the available light intensity (LI) to predict biomass productivity and evaluate overall performance.

During the initial phase of the study, I integrated a membrane filter with a bench-top photobioreactor (PBR) and created a continuously operating system. Recycling permeate reduced the amount of fresh medium delivered to the PBR by 45%. Biomass production rates as high as 400 mg-DW/L/d (9.2 g-DW/m2/d) were sustained under constant lighting over a 12-day period.

In the next phase, I operated the system as a sequencing batch reactor (SBR), which improved control over nutrient delivery and increased the concentration factor of filtered biomass (from 1.8 to 6.8). I developed unique system parameters to compute the amount of recycled permeate in the reactor and the actual hydraulic retention time during SBR operation. The amount of medium delivered to the system was reduced by up to 80%, and growth rates were consistent at variable amounts of repeatedly recycled permeate. The light-based model accurately predicted growth when biofilm was not present. Coupled with mass ratios for PCC 6803, these predictions facilitated efficient delivery of nitrogen and phosphorus. Daily biomass production rates and specific growth rates equal to 360 mg-DW/L/d (8.3 g/m2/d) and 1.0 d-1, respectively, were consistently achieved at a relatively low incident LI (180 µE/m2/s). Higher productivities (up to 550 mg-DW/L/d) occurred under increased LI (725 µE/m2/s), although the onset of biofilm impeded modeled performance.

Permeate did not cause any gradual growth inhibition. Repeated results showed cultures rapidly entered a stressed state, which was followed by widespread cell lysis. This phenomenon occurred independently of permeate recycling and was not caused by nutrient starvation. It may best be explained by negative allelopathic effects or viral infection as a result of mixed culture conditions.
ContributorsThompson, Matthew (Author) / Rittmann, Bruce E. (Thesis advisor) / Fox, Peter (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2015
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Description
Uranium (U) contamination has been attracting public concern, and many researchers are investigating principles and applications of U remediation. The overall goal of my research is to understand the versatile roles of sulfate-reducing bacteria (SRB) in uranium bioremediation, including direct involvement (reducing U) and indirect involvement (protecting U reoxidation). I

Uranium (U) contamination has been attracting public concern, and many researchers are investigating principles and applications of U remediation. The overall goal of my research is to understand the versatile roles of sulfate-reducing bacteria (SRB) in uranium bioremediation, including direct involvement (reducing U) and indirect involvement (protecting U reoxidation). I pursue this goal by studying Desulfovibro vuglaris, a representative SRB. For direct involvement, I performed experiments on uranium bioreduction and uraninite (UO2) production in batch tests and in a H2-based membrane biofilm reactor (MBfR) inoculated with D. vuglaris. In summary, D. vuglaris was able to immobilize soluble U(VI) by enzymatically reducing it to insoluble U(IV), and the nanocrystallinte UO2 was associated with the biomass. In the MBfR system, although D. vuglaris failed to form a biofilm, other microbial groups capable of U(VI) reduction formed a biofilm, and up to 95% U removal was achieved during a long-term operation. For the indirect involvement, I studied the production and characterization of and biogenic iron sulfide (FeS) in batch tests. In summary, D. vuglaris produced nanocrystalline FeS, a potential redox buffer to protect UO2 from remobilization by O2. My results demonstrate that a variety of controllable environmental parameters, including pH, free sulfide, and types of Fe sources and electron donors, significantly determined the characteristics of both biogenic solids, and those characteristics should affect U-sequestrating performance by SRB. Overall, my results provide a baseline for exploiting effective and sustainable approaches to U bioremediation, including the application of the novel MBfR technology to U sequestration from groundwater and biogenic FeS for protecting remobilization of sequestrated U, as well as the microbe-relevant tools to optimize U sequestration applicable in reality.
ContributorsZhou, Chen (Author) / Rittmann, Bruce E. (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2014
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Description
The microbial electrochemical cell (MXC) is a novel environmental-biotechnology platform for renewable energy production from waste streams. The two main goals of MXCs are recovery of renewable energy and production of clean water. Up to now, energy recovery, Coulombic efficiency (CE), and treatment efficiency of MXCs fed with real wastewater

The microbial electrochemical cell (MXC) is a novel environmental-biotechnology platform for renewable energy production from waste streams. The two main goals of MXCs are recovery of renewable energy and production of clean water. Up to now, energy recovery, Coulombic efficiency (CE), and treatment efficiency of MXCs fed with real wastewater have been low. Therefore, the overarching goal of my research was to address the main causes for these low efficiencies; this knowledge will advance MXCs technology toward commercialization.

First, I found that fermentation, not anode respiration, was the rate-limiting step for achieving complete organics removal, along with high current densities and CE. The best performance was achieved by doing most of the fermentation in an independent reactor that preceded the MXC. I also outlined how the efficiency of fermentation inside MXCs can be enhanced in order to make MXCs-based technologies cost-competitive with other anaerobic environmental biotechnologies. I revealed that the carbohydrate and protein contents and the BOD5/COD ratio governed the efficiency of organic-matter fermentation: high protein content and low BOD5/COD ratio were the main causes for low fermentation efficiency.

Next, I showed how a high ammonium concentration can provide kinetic and metabolic advantages or disadvantages for anode-respiring bacteria (ARB) over their competitors, particularly methanogens. When exposed to a relatively high ammonium concentration (i.e., > 2.2 g total ammonia-nitrogen (TAN)/L), the ARB were forced to divert a greater electron flow toward current generation and, consequently, had lower net biomass yield. However, the ARB were relatively more resistant to high free ammonia-nitrogen (FAN) concentrations, up to 200 mg FAN/L. I used FAN to manage ecological interactions among ARB and non-ARB in an MXC fed with fermentable substrate (glucose). Utilizing a combination of chemical, electrochemical, and genomic tools, I found that increased FAN led to higher CE and lower methane (CH4) production by suppressing methanogens. Thus, managing FAN offers a practical means to suppress methanogenesis, instead of using expensive and unrealistic inhibitors. My research findings open up new opportunities for more efficient operation of MXCs; this will enhance MXC scale-up and commercial applications, particularly for energy-positive treatment of waste streams containing recalcitrant organics.
ContributorsMohamed, Mohamed Mahmoud Ali (Author) / Rittmann, Bruce E. (Thesis advisor) / Torres, Cesar I. (Thesis advisor) / Westerhoff, Paul (Committee member) / Parameswaran, Prathap (Committee member) / Arizona State University (Publisher)
Created2016
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Description
ABSTRACT

Sustainable global energy production is one of the grand challenges of the 21st century. Next-generation renewable energy sources include using photosynthetic microbes such as cyanobacteria for efficient production of sustainable fuels from sunlight. The cyanobacterium Synechocystis PCC 6803 (Synechocystis) is a genetically tractable model organism for plant-like photosynthesis that is

ABSTRACT

Sustainable global energy production is one of the grand challenges of the 21st century. Next-generation renewable energy sources include using photosynthetic microbes such as cyanobacteria for efficient production of sustainable fuels from sunlight. The cyanobacterium Synechocystis PCC 6803 (Synechocystis) is a genetically tractable model organism for plant-like photosynthesis that is used to develop microbial biofuel technologies. However, outside of photosynthetic processes, relatively little is known about the biology of microbial phototrophs such as Synechocystis, which impairs their development into market-ready technologies. My research objective was to characterize strategic aspects of Synechocystis biology related to its use in biofuel production; specifically, how the cell surface modulates the interactions between Synechocystis cells and the environment. First, I documented extensive biofouling, or unwanted biofilm formation, in a 4,000-liter roof-top photobioreactor (PBR) used to cultivate Synechocystis, and correlated this cell-binding phenotype with changes in nutrient status by developing a bench-scale assay for axenic phototrophic biofilm formation. Second, I created a library of mutants that lack cell surface structures, and used this biofilm assay to show that mutants lacking the structures pili or S-layer have a non-biofouling phenotype. Third, I analyzed the transcriptomes of cultures showing aggregation, another cell-binding phenotype, and demonstrated that the cells were undergoing stringent response, a type of conserved stress response. Finally, I used contaminant Consortia and statistical modeling to test whether Synechocystis mutants lacking cell surface structures could reduce contaminant growth in mixed cultures. In summary, I have identified genetic and environmental means of manipulating Synechocystis strains for customized adhesion phenotypes, for more economical biomass harvesting and non-biofouling methods. Additionally, I developed a modified biofilm assay and demonstrated its utility in closing a key gap in the field of microbiology related to axenic phototrophic biofilm formation assays. Also, I demonstrated that statistical modeling of contaminant Consortia predicts contaminant growth across diverse species. Collectively, these findings serve as the basis for immediately lowering the cost barrier of Synechocystis biofuels via a more economical biomass-dewatering step, and provide new research tools for improving Synechocystis strains and culture ecology management for improved biofuel production.
ContributorsAllen, Rebecca Custer (Author) / Curtiss Iii, Roy (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Rittmann, Bruce E. (Committee member) / Vermaas, Willem (Committee member) / Arizona State University (Publisher)
Created2016
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Description
One solution to mitigating global climate change is using cyanobacteria or single-celled algae (collectively microalgae) to replace petroleum-based fuels and products, thereby reducing the net release of carbon dioxide. This work develops and evaluates a mechanistic kinetic model for light-dependent microalgal growth. Light interacts with microalgae in a

One solution to mitigating global climate change is using cyanobacteria or single-celled algae (collectively microalgae) to replace petroleum-based fuels and products, thereby reducing the net release of carbon dioxide. This work develops and evaluates a mechanistic kinetic model for light-dependent microalgal growth. Light interacts with microalgae in a variety of positive and negative ways that are captured by the model: light intensity (LI) attenuates through a microalgal culture, light absorption provides the energy and electron flows that drive photosynthesis, microalgae pool absorbed light energy, microalgae acclimate to different LI conditions, too-high LI causes damage to the cells’ photosystems, and sharp increases in light cause severe photoinhibition that inhibits growth. The model accounts for all these phenomena by using a set of state variables that represent the pooled light energy, photoacclimation, PSII photo-damage, PSII repair inhibition and PSI photodamage. Sets of experiments were conducted with the cyanobacterium Synechocystis sp. PCC 6803 during step-changes in light intensity and flashing light. The model was able to represent and explain all phenomena observed in the experiments. This included the spike and depression in growth rate following an increasing light step, the temporary depression in growth rate following a decreasing light step, the shape of the steady-state growth-irradiance curve, and the “blending” of light and dark periods under rapid flashes of light. The LI model is a marked improvement over previous light-dependent growth models, and can be used to design and interpret future experiments and practical systems for generating renewable feedstock to replace petroleum.
ContributorsStraka, Levi (Author) / Rittmann, Bruce E. (Thesis advisor) / Fox, Peter (Committee member) / Torres, César I (Committee member) / Arizona State University (Publisher)
Created2017
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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

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
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Description
Eighty-two percent of the United States population reside in urban areas. The centralized treatment of the municipal wastewater produced by this population is a huge energy expenditure, up to three percent of the entire energy budget of the country. A portion of this energy is able to be recovered

Eighty-two percent of the United States population reside in urban areas. The centralized treatment of the municipal wastewater produced by this population is a huge energy expenditure, up to three percent of the entire energy budget of the country. A portion of this energy is able to be recovered through the process of anaerobic sludge digestion. Typically, this technology converts the solids separated and generated during the wastewater treatment process into methane, a combustible gas that may be burned to generate electricity. Designing and optimizing anaerobic digestion systems requires the measurement of degradation rates for waste-specific kinetic parameters. In this work, I discuss the ways these kinetic parameters are typically measured. I recommend and demonstrate improvements to these commonly used measuring techniques. I provide experimental results of batch kinetic experiments exploring the effect of sludge pretreatment, a process designed to facilitate rapid breakdown of recalcitrant solids, on energy recovery rates. I explore the use of microbial electrochemical cells, an alternative energy recovery technology able to produce electricity directly from sludge digestion, as precise reporters of degradation kinetics. Finally, I examine a fundamental kinetic limitation of microbial electrochemical cells, acidification of the anode respiring biofilm, to improve their performance as kinetic sensors or energy recovery technologies.
ContributorsHart, Steven Gregg (Author) / Torres, César I (Thesis advisor) / Parameswaran, Prathap (Committee member) / Rittmann, Bruce E. (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2020
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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

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.
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
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Description
Electro-Selective Fermentation (ESF) combines Selective Fermentation (SF) and a Microbial Electrolysis Cell (MEC) to selectively degrade carbohydrate and protein in lipid-rich microalgae biomass, enhancing lipid wet-extraction. In addition, saturated long-chain fatty acids (LCFAs) are produced via β-oxidation. This dissertation builds understanding of the biochemical phenomena and microbial interactions

Electro-Selective Fermentation (ESF) combines Selective Fermentation (SF) and a Microbial Electrolysis Cell (MEC) to selectively degrade carbohydrate and protein in lipid-rich microalgae biomass, enhancing lipid wet-extraction. In addition, saturated long-chain fatty acids (LCFAs) are produced via β-oxidation. This dissertation builds understanding of the biochemical phenomena and microbial interactions occurring among fermenters, lipid biohydrogenaters, and anode respiring bacteria (ARB) in ESF. The work begins by proving that ESF is effective in enhancing lipid wet-extraction from Scenedesmus acutus biomass, while also achieving “biohydrogenation” to produce saturated LCFAs. Increasing anode respiration effectively scavenges short chain fatty acids (SCFAs) generated by fermentation, reducing electron loss. However, the effectiveness of ESF depends on biochemical characteristics of the feeding biomass (FB). Four different FB batches yield different lipid-extraction performances, based on the composition of FB’s cellular structure. Finally, starting an ESF reactor with a long solid retention time (SRT), but then switching it to a short SRT provides high lipid extractability and volumetric production with low lipid los. Lipid fermenters can be flushed out with short a SRT, but starting with a short SRT fails achieve good results because fermenters needed to degrading algal protective layers also are flushed out and fail to recover when a long SRT is imposed. These results point to a potentially useful technology to harvest lipid from microalgae, as well as insight about how this technology can be best managed.
ContributorsLiu, Yuanzhen (Author) / Rittmann, Bruce E. (Thesis advisor) / Torres, César I (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2019
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Description
Earthquake-induced soil liquefaction poses a significant global threat, especially to vulnerable populations. There are no existing cost-effective techniques for mitigation of liquefaction under or around existing infrastructure. Microbially Induced Desaturation and Precipitation (MIDP) via denitrification is a potentially sustainable, non-disruptive bio-based ground improvement technique under existing structures. MIDP

Earthquake-induced soil liquefaction poses a significant global threat, especially to vulnerable populations. There are no existing cost-effective techniques for mitigation of liquefaction under or around existing infrastructure. Microbially Induced Desaturation and Precipitation (MIDP) via denitrification is a potentially sustainable, non-disruptive bio-based ground improvement technique under existing structures. MIDP has been shown to reduce liquefaction triggering potential under lab conditions in two ways: 1) biogenic gas desaturation in the short-term (treatment within hours to days) and 2) calcium carbonate precipitation and soil strengthening in the long-term (treatment within weeks to months). However, these experiments have not considered MIDP behavior under field stresses and pressures, nor have they considered challenges from process inhibition or microbial competition that may be encountered when upscaled to field applications. This study presents results from centrifuge experiments and simplified modeling to explore scaling effects on biogenic gas formation, distribution, and retention when simulating field pressures and stresses. Experimental results from the centrifuge demonstrated MIDP’s potential to mitigate the potential liquefaction triggering through desaturation. This study also includes the development of a biogeochemical model to explore the impact of water constituents, process inhibition, and alternative biochemical metabolisms on MIDP and the subsequent impact of MIDP on the surrounding environment. The model was used to explore MIDP behavior when varying the source-water used as the substrate recipe solute (i.e., groundwater and seawater) and when varying the electron donor (i.e., acetate, glucose, and molasses) in different substrate recipes. The predicted products and by-products were compared for cases when desaturation was the targeted improvement mechanism and for the case when precipitation was the primary targeted ground improvement mechanism. From these modeling exercises, MIDP can be applied in all tested natural environments and adjusting the substrate recipe may be able to mitigate unwanted long-term environmental impacts. A preliminary techno-economic analysis using information gained from the modeling exercises was performed, which demonstrated MIDP’s potential as a cost-effective technique compared to currently used ground improvement techniques, which can be costly, impractical, and unsustainable. The findings from this study are critical to develop treatment MIDP plans for potential field trials to maximize treatment effectiveness, promote sustainability and cost-effectiveness, and limit unwanted by-products.
ContributorsHall, Caitlyn Anne (Author) / Rittmann, Bruce E. (Thesis advisor) / Kavazanjian, Edward (Thesis advisor) / van Paassen, Leon A. (Committee member) / DeJong, Jason T. (Committee member) / Arizona State University (Publisher)
Created2021