Mountain Scholar :: Browsing by Author "Peebles, Christie, committee member"

Browsing by Author "Peebles, Christie, committee member"

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Open Access
Archaeal isoprenoid biosynthesis
(Colorado State University. Libraries, 2018) Liman, Lie Stefanus Geraldy, author; Santangelo, Thomas, advisor; Laybourn, Paul, committee member; Peebles, Christie, committee member
Many high value natural products - including artemisinin, squalene, and farnesene – are isoprenoids. Efforts to commercially produce isoprenoids are often complicated by low concentrations of isoprenoid precursors and the toxicity of isoprenoids in common production platforms (i.e. bacteria and yeasts). Archaeal-based production platforms provide a potential solution to the precursor toxicity problems as archaea produce isoprenoids in large quantities to generate their unique membrane hydrocarbon chains. One roadblock to commercial archaeal isoprenoid production platforms is the uncharacterized pathway leading to isoprenoid precursor synthesis. This project details, genetically and biochemical, the first three steps in the proposed pathway of archaeal isoprenoid biosynthesis - from acetyl-CoA to mevalonate - in Thermococcus kodakarensis.
Open Access
Bioconversion of lipid-extracted algal biomass into ethanol
(Colorado State University. Libraries, 2016) Mirsiaghi, Mona, author; Reardon, Kenneth F., advisor; Peebles, Christie, committee member; Peers, Graham, committee member; Smith, Gordon, committee member
Energy security, high atmospheric greenhouse gas levels, and issues associated with fossil fuel extraction are among the incentives for developing alternative and renewable energy resources. Biofuels, produced from a wide range of feedstocks, have the potential to reduce greenhouse gas emissions. In particular, the use of microalgae as a feedstock has received a high level of interest in recent years. Microalgal biofuels are promising replacement for fossil fuels and have the potential to displace petroleum-based fuels while decrease greenhouse gas emissions. The primary focus of research and development toward algal biofuels has been on the production of biodiesel or renewable diesel from the lipid fraction, with use of the non-lipid biomass fraction for production of biogas, electricity, animal feed, or fertilizer. Since the non-lipid fraction, consisting of mainly carbohydrates and proteins, comprises approximately half of the algal biomass, our approach is biological conversion of the lipid-extracted algal biomass (LEAB) into fuels. We used LEAB from Nannochloropsis salina, and ethanol was the model product. The first step in conversion of LEAB to ethanol was deconstruction of the cell wall into fermentable substrates by using different acids or enzymes. Sugar release yields and rates were compared for different treatments. One-step sulfuric acid hydrolysis had the highest yield of released sugars, while the one-step hydrochloric acid treatment had the highest sugar release rate. Enzymatic hydrolysis produced acceptable sugar release rates and yields but enzymes designed for algal biomass deconstruction are still needed. Proteins were deconstructed using a commercially available protease. The hydrolysate, containing the released sugars, peptides, and amino acids, was used as a fermentation medium with no added nutrients. Three ethanologenic microorganisms were used for fermentation: two strains of Saccharomyces cerevisiae (JAY270 and ATCC 26603) and Zymomonas mobilis ATCC 10988. Ethanol yields and productivities were compared. Among the studied microorganisms, JAY270 had the highest ethanol yield while Z. mobilis had the lowest yield for most of the studied conditions. A protease treatment improved the biomass and ethanol yields of JAY270 by providing more carbon and nitrogen. To increase ethanol productivity, a continuous fermentation approach was adapted. Continuous stirred tank reactors have increased productivity over batch systems due to lower idle time. The downtime associated with batch fermentation is the time it takes for empting, cleaning, and filling the reactor. Productivity in the continuous fermentation was limited by the growth characteristics of the microorganism since at high flow rates, with washout occurring below a critical residence time. To overcome the washout problem, the use of an immobilized cell reactor was explored. The performance (ethanol productivity) of free and immobilized cells was compared using an enzymatic hydrolysate of LEAB. Higher ethanol productivities were observed for the continuous immobilized cell reactor compared to the stirred tank reactor.
Open Access
"Biofilmomics": functional protein expression in biofilm biotechnologies revealed by quantitative proteomics
(Colorado State University. Libraries, 2020) Chignell, Jeremy, author; Reardon, Kenneth, advisor; De Long, Susan, advisor; Peebles, Christie, committee member; Sharvelle, Sybil, committee member
Microbial biotechnologies that utilize biofilms often exhibit superior performance compared with planktonic systems. Many details of biofilm metabolism that drive those improvements in performance remain unclear. Only recently have molecular tools emerged that can provide a holistic picture of life in a complex biosystem like a biofilm for the purposes of answering questions on a system level. The purpose of this work was to address four fundamental questions about protein expression in biofilms: what kind of protein expression is distinctive to biofilms? Which biofilm proteins are associated with a function of interest? How does co-culture with another species affect biofilm-related protein expression? When during multi-species biofilm development does a function of interest emerge and who in the community is responsible? Label-free quantitative proteomics was used in conjunction with physiological experimentation to address these four questions. In the first study we found that L. delbrueckii lactis protein expression in flow-cell biofilms was 31% more diverse than in planktonic cultures, and proteins related to catalytic activity were significantly increased in biofilms at the expense of proteins for cell motility and replication. Roles for riboflavin and fatty acid metabolism suggested modulations in redox functions and membrane turnover during life in a biofilm. The second study compared protein expression by S. onedensis MR-1 in electricity-generating biofilms with that in aerobic biofilms from the same microbial fuel cell reactor. Three novel proteins associated with electricity generation were identified, in addition to proteomic evidence of aerobic metabolism by anode biofilm cells. The latter result was shown to be consistent with kinetics of oxygen depletion and bulk cell growth in the MFC, suggesting operational conditions to reduce this bulk cell growth and thereby reduce fouling of the cathode and improve overall Coulombic efficiency of the single-chamber MFC system. In the third study, it was discovered through proteomic and physiological experiments that a virulent phenotype associated with biofilm formation was triggered in P. putida when co-cultured with B. atrophaeus. Dramatic shifts in protein expression at the initial trigger point of virulent biofilm formation by P. putida are described. Finally, a comparison of the meta-proteomes of microbial fuel cell biofilms at different stages of development indicated that proteins in metabolic pathways for carbon storage and competitive inhibition are differentially expressed when the biofilm becomes electrochemically active. Meta-proteomics and 16S rRNA gene sequencing agreed that it is possible for a microbial fuel cell community to maintain high diversity (and therefore potentially higher resilience) while generating electricity at levels comparable to a MFC community dominated by Geobacter. Each of these chapters was prepared as an independent manuscript, though the themes were integrated by the overall theme of quantifying differential protein expression in biofilms in order to reveal new details about their development and functionality. Since the performance of many engineered biosystems—including those that employ biofilms—often can be controlled adequately at an operational level, an attitude persists that any additional molecular investigation is superfluous. The work presented here provides evidence for the opposite viewpoint: a rich understanding of the molecular mechanisms behind biofilm functionality can inform strategies for continuous system improvement and suggest new capabilities and biotechnological applications of biofilms.
Open Access
Development of a phage-based diagnostic sensor for active Tuberculosis
(Colorado State University. Libraries, 2016) Zhao, Ning, author; Fisk, John D., advisor; Spencer, John S., committee member; Henry, Charles S., committee member; Peebles, Christie, committee member
Antibodies, the quintessential biological recognition molecules, are not ideal for many applications because of their large size, complex modifications, and thermal and chemical instability. Identifying alternative scaffolds that can be evolved into tight, specific binding molecules with improved physical properties is of increasing interest, particularly for biomedical applications in resource-limited environments. Hyperthermophilic organisms, such as Sulfolobus solfataricus, are an attractive source of highly stable proteins as starting points for alternative molecular recognition scaffolds. We describe the first application of phage display to identify binding proteins based on the Sulfolobus solfataricus protein Sso7d scaffold. Sso7d is a small (approximately 7 kDa, 63 amino acids), cysteine free DNA-binding protein with a melting temperature of nearly 100 °C. Tight binding Sso7d variants were selected for a diverse set of protein targets from a 1010 member library, demonstrating the versatility of the scaffold. These Sso7d variants are able to discriminate among closely-related human, bovine, and rabbit serum albumins. Equilibrium dissociation constants in the nanomolar to low micromolar range were measured via competitive ELISA. Importantly, the Sso7d variants continue to bind their targets in the absence of the phage context. Furthermore, phage-displayed Sso7d variants retain their binding affinity after exposure to temperatures up to 70 °C. Taken together, our results suggest that the Sso7d scaffold will be a complementary addition to the range of non-antibody scaffold proteins that can be utilized in phage display. Variants of hyperthermostable binding proteins have potential applications in diagnostics and therapeutics for environments with extreme conditions of storage and deployment. One application for utilizing Sso7d evolved binding molecules is development of Tuberculosis (TB) diagnostic tests. TB is the leading cause of death from infectious disease worldwide. The low sensitivity, extended processing time and high expense of diagnostics are major challenges to the detection and treatment of TB. Mycobacterium tuberculosis ornithine transcarbamylase (Mtb OTC, Rv1656) has been identified in the urine of patients with active TB infection, making Mtb OTC a promising target for point-of-care diagnostics in resource-limited settings. We are motivated to engineer phage-based diagnostic systems that feature improved physical stability, cost of production and sensitivity relative to traditional antibody-based reagents. Specific binding proteins with low nanomolar affinities for Mtb OTC were selected from the naïve Sso7d phage library. Phage particles displaying Sso7d variants along with a monoclonal antibody (mAb) generated by hybridoma technology were utilized to generate a capture ELISA-based assay for Mtb OTC. The ELISA assay signal is linear over the target concentration range of 2.0-125.0 ng/mL with limit of detection 0.4 ng/mL (12 pM), which is comparable to commercial available antibody-based assays. Importantly, this assay maintains functionality at both neutral and basic pH in presence of salt and urea over the range of concentrations typical for human urine. Furthermore, towards our phage-based diagnostic test development goal, a test with a pair of phage displaying 2 different Sso7d variants was established with a limit of detection 4.5 ng/mL (130 pM). Stability of TB diagnostic test is improved at acidic conditions in presence of salt and urea in the typical concentration range of human urine, which may due to the replacement of mAb with phage particles. This result demonstrates that phage particles replacing antibodies in the diagnostic test has the potential to improve stability at harsh conditions.
Open Access
Directed expression of R2R3 MYB transcription factors for ectopic suberin deposition
(Colorado State University. Libraries, 2022) McKay Whiteman, Angel, author; Medford, June, advisor; Nishimura, Marc, committee member; Peebles, Christie, committee member
Plants are susceptible to many stresses, which can lead to reduced growth, decreased crop yield, or even plant death. Plants contain natural barriers to protect them from stresses, including the hydrophobic biopolymer suberin. Suberin protects plants against a variety of factors, including water loss, toxic ions, loss of essential nutrients, and entry of microorganisms. Plant roots contain a suberized barrier called the endodermis, which regulates the entry and exit of materials from the cortex to the vascular cylinder. While the endodermis protects the vascular cylinder, the cortex is left susceptible to stresses. Engineering a suberized barrier in the root epidermis could protect the cortex and provide an additional point of regulation. Multiple R2R3 type MYB transcription factors have been found to be involved in suberin biosynthesis. Expressing these transcription factors in the root epidermis could provide a suberin barrier for protection. I expressed five R2R3 MYB transcription factors in the root epidermis of Arabidopsis thaliana to determine whether their expression led to suberization of the root epidermis. Constitutive expression of one transcription factor, MYB84, led to increased epidermal suberin. Two homozygous lines were further analyzed and found to have decreased root growth. Gene expression results from one homozygous line suggest that MYB84 overexpression may lead to increased expression of suberin biosynthetic genes involved in synthesis of aliphatic suberin monomers and monomer transport. Further analysis of these transgenic plants could provide insight into the potential protective barrier the root epidermal suberin provides.
Open Access
Ectopic expression of R2R3-MYB transcription factors to control suberin biosynthesis
(Colorado State University. Libraries, 2022) Berning, Nick, author; Medford, June, advisor; Peebles, Christie, committee member; Sloan, Dan, committee member
Minimizing the deleterious effects of abiotic stresses on cultivated plants is critical to maximizing crop yield. Suberin is a glycerol based polyester found in the endodermis, seed coat, cork cells, and areas of wounding in the epidermis. Recently, suberin biosynthesis has been shown to be at least partially regulated by a set of R2R3-MYB transcription factors. The ability to control suberin biosynthesis in specific plant tissues could be a valuable biotechnological tool in designing plants which can withstand higher degrees of abiotic stress. In this thesis, I detail a genetic screen of four different R2R3-MYB transcription factor's ability to induce ectopic suberin formation in the root epidermis of Arabidopsis thaliana. Subsequently, I characterize the transcription factor with the greatest ability to induce ectopic suberin biosynthesis, MYB92. MYB92, when expressed in the root epidermis, consistently forms a suberin barrier within that tissue. Plants expressing MYB92 in the root epidermis may be stunted and chlorotic under typical growth conditions, however, they outperform wild-type Col-0 plants under salt stress. More characterization of ectopic, suberin barrier's ability to confer salt tolerance could be performed in order to understand how epidermal suberin might perform in crop plants.
Open Access
Engineering stabilized enzymes via computational design and immobilization
(Colorado State University. Libraries, 2016) Johnson, Lucas B., author; Snow, Christopher, advisor; Reardon, Kenneth, committee member; Peebles, Christie, committee member; Peersen, Olve, committee member
The realm of biocatalysis has significantly matured beyond ancient fermentation techniques to accommodate the demand for modern day products. Enzymatically produced goods already influence our daily lives, from sweeteners and laundry detergent to blood pressure medication and antibiotics. Protein engineering has been a major driving force behind this biorevolution, yielding catalysts that can transform non-native substrates and withstand harsh industrial conditions. Although successful in many regards, computational design efforts are still limited by the crude approximations employed in searching a complex energy landscape. Advancements in protein engineering methods will be necessary to develop our understanding of biomolecules and accelerate the next generation of biotechnology applications. Our work employs a combination of computational design and simulation to achieve improved enzyme stability. In the first example, an enzyme used in the production of cellulosic biofuels was redesigned to remain active at high temperature. An initial approach involving consensus sequence analysis, predicted point mutation energy, and combinatorial optimization resulted in a sequence with reduced stability and activity. However, by using recombination methods and molecular dynamics simulations, we were able to identify specific mutations that had a stabilizing or destabilizing effect, and we successfully isolated mutations that benefited enzyme stability. Our iterative approach demonstrated how common design failures could be overcome by careful interpretation and suggested methods for improving future computational design efforts. In the second example, a cellulase was designed to have a high net charge via selected surface mutagenesis. “Supercharged” cellulases were experimentally characterized in various ionic liquids to assess the effect of high ion concentration on enzyme stability and activity. The designed enzymes also provided an opportunity to systematically probe the protein-solvent interface. Molecular dynamics simulations showed how ions influenced protein behavior by inducing minor unfolding events or by physically blocking the active site. Contradictory to previous reports, charged mutations only appeared to alter the affinity of anions and did not significantly change the binding of cations at the protein surface. Understanding the different modes of enzyme inactivation could motivate targeted design strategies for engineering protein resilience in ionic solvents. In addition to the discussed computational design methods, immobilization strategies were identified for capturing enzymes within porous protein crystals. Immobilization offers a generic approach for improving enzyme stability and activity. Our preliminary studies involving horseradish peroxidase and other enzymes suggested protein scaffolds could be employed as an effective immobilization material. Co-immobilizing multiple enzymes within the porous material led to improved product yield via exclusion of off-pathway reactions. Although future studies will be required to assess the potential capabilities of this immobilization strategy in comparison to other materials, preliminary results suggest protein crystals offer a favorable, controlled environment for immobilizing enzymes. The diversity of approaches presented in this thesis emphasizes that there are many options for engineering enzyme stability. Extending the lessons learned from our cellulase engineering to the greater field of rational protein design promotes the concept of biomolecules as designable entities. By establishing the shortcomings of our designs and suggesting routes for improvement, we anticipate our design methods and immobilization strategies will procure continued interest from the biotechnology community. The toolsets we developed for cellulases can be directly transferred to other enzymes and have the potential to impact a range of protein engineering applications.
Open Access
Establishment and systematic characterization of Mycobacterium tuberculosis in bioreactors
(Colorado State University. Libraries, 2016) Knabenbauer, Phillip, author; Dobos, Karen, advisor; Slayden, Richard, committee member; McNeil, Michael, committee member; Peebles, Christie, committee member
Mycobacterium tuberculosis infection is characterized by active and latent disease states. Granuloma-induced oxygen tension may shift bacteria into bacteriostatic persistence. Current models of hypoxia-induced mycobacteria have limitations, requiring establishment of novel culturing methods. Here, M. tuberculosis was propagated under defined oxygen concentration in bioreactors. Initial analyses confirmed mycobacterial non-replicating persistence. This study will provide insight into core physiological adaptations of M. tuberculosis while reducing bias from the contaminants during adaptation into dormancy. Here we describe a novel method of propagation using defined oxygen concentrations, then enrich the final culture for viability to remove transcriptional bias, and finally interrogate the presence of viable but non-culturable tubercle bacilli in order to obtain a greater sense of true viability. The current study will further contribute to our understanding of the physical adaptation of Mtb during growth and dormancy, by removing bias from the contaminating transcriptome gradient generated by the temporal adaptation of M. tuberculosis into dormancy. This will enhance the accuracy of downstream structural and transcriptomic analyses as well as give rise to a novel high throughput approach to M. tuberculosis propagation for research materials.
Open Access
Geographically-resolved evaluation of economic and environmental services from renewable diesel derived from attached algae flow-ways across the United States
(Colorado State University. Libraries, 2022) Banks, Austin Brice, author; Quinn, Jason, advisor; Peebles, Christie, committee member; Windom, Bret, committee member
Harmful algal blooms (HABs) are becoming more invasive and ever more prevalent due to rises in nitrogen and phosphorus pollution in watersheds. Nitrogen and phosphorus leakages primarily occur from non-point sources like agricultural runoff, but also point sources like wastewater treatment facilities. Previous efforts to reduce nitrogen and phosphorus loadings and mitigate HABs have largely been ineffective despite investment in nutrient reduction technologies. As the population grows, our consumption and dispersal of nitrogen and phosphorus is expected to compound, and HABs will continue to wreak havoc on our aquatic ecosystems. Herein, we introduce a novel biorefinery that taps into the vast sources of nitrogen and phosphorus in watersheds while simultaneously producing biofuels. Contaminated water is diverted to flow over attached algae systems, feeding native, periphytic algal cultures and scrubbing excessive nutrients from the water. Hydrothermal liquefaction converts the algal biomass into renewable fuels, nutrient-rich fertilizers, and carbonaceous char. The evaluation of the biorefinery concept is done through integrating geographically-resolved growth modeling with nutrient resource availability based on all Hydrologic Unit Code-8 (HUC8) in the contiguous US which is integrated into sustainability models to evaluate the economic and environmental impact of the proposed system. Life cycle analysis results demonstrate a global warming potential of 25 g CO2-eq MJ-1, a eutrophication potential of 1.3*10-5 kg N eq MJ-1, and a net energy ratio 0.33 of MJ MJ-1 in the Santa Monica Bay, CA subbasin. Technoeconomic assessments found that renewable diesel can be produced for $1.20 per cubic decimeter (dm-3) or $4.56 per gallon of gasoline equivalent (GGE-1) under optimal conditions in the Santa Monica Bay, CA subbasin, with results dramatically varying across the US. Water quality trading was also incorporated into the analysis. Using modest nutrient credit values of $4.5 per kg of total nitrogen (kg-TN-1) and $4.5 per kg total phosphorus (kg-TP-1) removed enabled the renewable diesel to achieve parity with conventional diesel, $1.01 dm-3 ($3.84 GGE-1) in the Santa Monica Bay, CA subbasin. A more aggressive credit value of $45 kg-TN-1 and $45 kg-TP-1 made the price of the renewable diesel negative in Santa Monica Bay, CA, roughly $-4.45 dm-3 ($-16.8 GGE-1), and across the Midwest, the Gulf of Mexico, and major cities on the East and West Coast. This means the value of the service that the algae provide in remediating watersheds covers all costs of the system to the point where the renewable diesel represents a product with negligible value. These results highlight a path forward for mitigating eutrophication while also creating a sustainable fuel. Discussion focuses on the service that large-scale deployment of attached algae flow-ways provide to remediate excessive nutrients from watersheds and generate biofuels at a cost-effective price point when water quality trading credits are incorporated into the system economics.
Open Access
Growth, recovery and bioaccumulation of alfalfa (Medicago sativa) and spinach (Spinacia oleracea) exposed to cyanotoxins in agricultural environments
(Colorado State University. Libraries, 2020) Nezat, Caryn Janel, author; Omur-Ozbek, Pinar, advisor; Peebles, Christie, committee member; Bailey, Ryan, committee member
Harmful algal blooms (HABs) are a growing concern for surface water resources around the globe. With increasing pressure on our limited fresh water resources due to climate change, the risk of contamination from HABs and the cyanobacterial toxins that accompany blooms, exacerbates the problem. Adverse health effects from cyanotoxin exposure has been documented in human and animal mortality and morbidity cases worldwide. Nationally, the presence and severity of HABs has prompted multiple cyanotoxins, including cylindrospermopsin (CYN) and microcystins (MCLR), to be listed on the USEPA Drinking Water Contaminant Candidate List-4 (CCL4) requiring many public systems to monitor for cyanotoxin presence. Recognizing this risk, the World Health Organization (WHO) has long established guidelines to acceptable levels in surface waters based on exposure pathways and use. Further concerns have arisen as our understanding about cyanotoxins has been expanded by research. The purpose of this experiment was to determine 1) effects of toxin exposure during germination, 2) the effects of CYN and MCLR on agricultural crops exposed to toxins during vegetative and mature growth stages, 3) crops ability to recover from toxin exposure and 4) to quantify amount of cyanotoxin accumulated within crop tissue after exposure to cyanotoxins. Germination results indicated exposure to CYN and MCLR did not decrease the rate of germination of alfalfa or spinach. Further, alfalfa and spinach had increased primary root growth for seeds exposed to cyanotoxins. During early vegetative exposure, spinach showed increased biomass and larger leaf area when exposed to MCLR and CYN. After a recovery period spinach plants exposed to CYN showed increased biomass compared to controls. Alfalfa plants exposed to MCLR in vegetative stages had significantly more biomass when compared to controls and this trend was observed after the recovery period. Results of alfalfa exposed during mature growth stages to CYN and MCLR indicated it was more sensitive to CYN, however both toxin treatments resulted in increased biomass production. After one- and two-weeks of recovery the MCLR treated alfalfa biomass remained higher than controls. Bioaccumulation of CYN and MCLR was observed in alfalfa exposed early to the toxins and detectable levels were observed after the one-week recovery period. Spinach accumulated MCLR during early exposures and had detectable levels in the stems after one-month recovery. During mature exposure, alfalfa initially only had detectable levels of MCLR, which decreased over the recovery periods. However, the presence of CYN was not detected until one-week prior to the final toxin exposure. These findings support the growing concern that use of cyanotoxin contaminated irrigation water can be an additional exposure route for ingestion of toxins and increased risk of adverse health effects. Further studies into the subsurface fate of cyanotoxins will further increase the understanding of their bioavailability and persistence in soil.
Open Access
Implications of cell composition and size on the performance of microalgae ultrasonic harvesting
(Colorado State University. Libraries, 2018) Aligata, Alyssa Jean, author; Marchese, Anthony, advisor; Quinn, Jason, advisor; Peebles, Christie, committee member
Substantial economic challenges exist across the value chain for microalgae-based biofuels and bioproducts. Acoustic harvesting could dramatically reduce harvesting costs and directly address current energy barriers to separating algae from growth media. This technology utilizes ultrasonic standing waves to create an acoustic radiation force that, due to differences in the acoustic properties of the cells and media, causes the microalgae cells to agglomerate and settle out of the solution. The magnitude of the acoustic radiation force is directly related to the cell radius and acoustic contrast factor (ACF), the latter of which is a function of the density and compressibility of the cell. These properties can vary widely depending on the algae species, cultivation conditions, and growth stage—all of which affect the composition of the microalgae cells (e.g., lipid, carbohydrate, protein content). In this work, two methods were used to determine the ACF of microalgal cells: 1) a property measurement approach and 2) a particle tracking approach. The first method involved experimentally measuring the size distribution, density and compressibility of the cells and calculating the ACF. The second method utilized particle tracking velocimetry and a COMSOL Multiphysics model to estimate the ACF. The ACF was characterized, using both techniques, for three species—Chlamydomonas reinhardtii, Nannochloropsis salina, and Tetraselmis chuii—as a function of dynamic cellular composition over a 2-week growth period. For C. reinhardtii the lipid content increased from 26% ± 1% to 40% ± 1% from day 3 to 9, which resulted in a 43% decrease in ACF (0.056 ± 0.003 to 0.032 ± 0.001). For N. salina the lipid content increased from 25% ± 1% to 33% ± 1% from day 3 to 10, which also resulted in a 43% decrease in ACF (0.040 ± 0.002 to 0.023 ± 0.001). For T. chuii the lipid content remained relatively stable (~10%) throughout the growth period so the ACF (~0.3) did not change significantly. ACF decreases as lipid content increases because lipids have a negative ACF in growth media, whereas carbohydrates and proteins have a positive ACF. However, cell size can have a greater impact on an algal strains' responsiveness to acoustic harvesting because the net force is proportional to Φa2. Furthermore, acoustic harvesting works best for large diameter cells, provided that those cells have a nonzero ACF. T. chuii had the largest cell diameter of approximately 12 µm, while C. reinhardtii and N. salina had cell diameters of 8.5 µm and 4.3 µm, respectively. The Φa2 values for T. chuii were approximately 50× higher than the values for N. salina, which is largely due to T. chuii cells having a diameter that is 3× the diameter of N. salina cells. Composition also contributed to the higher Φa2 values for T. chuii since these cells were composed of mostly carbohydrates and had an ACF that was an order of magnitude higher than the ACF of N. salina. This research shows that acoustic harvesting has the potential to positively impact the algal biofuels value chain through the reduction of energy required for harvesting.
Open Access
Interrogating reactions of gold nanoclusters: insights into catalysis and the Brust-Schiffrin synthesis
(Colorado State University. Libraries, 2017) Dreier, Timothy Andrew, author; Ackerson, Christopher J., advisor; Kennan, Alan J., committee member; Henry, Charles, committee member; Peebles, Christie, committee member
Over the past several decades, interest in the synthesis and behavior of atomically precise gold nanoclusters has gained substantial momentum. Herein, both catalytic behavior and synthetic mechanisms are explored using techniques more typically applied to organic chemistry. In the case of catalysis, Au25(SR)18 has emerged as a well-studied model system. In an effort to investigate their potential as intact, homogeneous, unsupported catalysts, we have discovered that Au25(SR)18 clusters are not stable in oxidizing conditions reported for catalytic styrene oxidation. Further investigation suggests that the active catalytic species is an Au(I) species resulting from oxidative decomposition of the starting gold cluster. Equally important to chemical behavior is an understanding of the reaction dynamics during the synthesis of atomically precise clusters. Because the Brust-Schiffrin method is the standard procedure by which gold nanoclusters are synthesized, the role of oxygen in it has been investigated for both organic and aqueous systems. In either case, it is clear obtaining the desired product depends on a radically mediated etching step. These results give new insight into how the Brust-Schiffrin method might be modified to further synthesis of uniquely interesting nanocluster systems.
Open Access
Investigation and applications of current and novel sustainability sciences
(Colorado State University. Libraries, 2020) DeRose, Katherine K., author; Quinn, Jason C., advisor; Marchese, Anthony J., committee member; Jathar, Shantanu, committee member; Peebles, Christie, committee member
Engineering-based sustainable solutions are required to ensure continued access to energy, food and clean water for a growing population. Techno-economic analysis and life cycle assessment provide a means of evaluating emerging technologies to determine if they can economically and sustainably provide solutions to current and future resource demands. Concurrently, performance targets can be identified to help drive technology forward in a sustainable fashion. A major advantage of sustainability analyses is the ability to perform early-stage technology evaluation prior to intensive research investment. The application of these techniques can be applied to a variety of technologies including renewable bio-based fuels. It is also necessary to understand the limitations of these sustainability sciences to properly interpret analysis results. This work focuses on the applications of sustainability sciences to multiple technologies including foundational investigation of the methodology behind assessments. The first technology evaluation showcases the iterative nature and relationship between sustainability sciences and research for a novel biofuel conversion process using algae as a feedstock. The second technology evaluation seeks to improve sustainability metrics of the widespread corn-ethanol process; and is also used as a case study to identify limitations in current sustainability sciences. The final technology evaluation is a novel application of sustainability sciences to identify technology solutions for environmental disruptions. Microalgae has been a feedstock of interest for renewable fuels production, but the technology remains impeded due to high growth costs. Most research has been focused on increasing biomass productivity and lipid content and/or reducing capital and operation costs associated with traditional growth systems. An alternative approach is to consider an entirely new growth method; attached-growth systems. Sustainability modeling was used to identify the optimal processing opportunities for the production of renewable fuels from algae grown in this method through the use of economic and environmental analyses. Results indicated that ash reduction, energy intensive processing and high growth costs needed to be addressed to improve economic viability. A secondary effort focused on advancing the research and modelling to further refine results based on this focus. Results show minimum fuel selling prices ranging between $9.13 to $31.22 per gallon of gasoline equivalent, dependent on scenario and process assumptions. Sustainability analyses can also be applied to improve current technologies. Corn ethanol represents a mature technology with a long production history as a first-generation alternative fuel but has been widely criticized for high production costs and only marginal sustainability improvements over traditional petroleum-based fuels. One approach for improving these metrics is to focus on increased utilization of co-products through additional processing. Sustainability analysis results indicate an additional co-product fermentation process may be considered as a value-add for refiners but is dependent on economic and product market assumptions. This process was also used as a case study to explore how life cycle methodology affects environmental impact results. Life cycle assessment (LCA) results have a broad variability with well-to-pump results ranging between 42 to 210 g CO2-eq MJ fuel-1, dependent on co-product allocation methodology. This variability within the results can affect a product's ability to meet environmental standards, such as the Renewable Fuel Standard, and represents a critical area for improved methodological guidance. In addition to technology, sustainability sciences can also be applied to identify economically viable solutions for environmental disruptions such as harmful algae blooms (HAB's). HAB's affect both fresh and saltwater bodies around the world, causing a variety of environmental and economic damages to surrounding ecosystems and communities. The primary driver of HAB's is eutrophication, or excess nutrients in the water, and the principal approach to mitigating HAB's is reducing nutrients before they collect en masse downstream. Technology solutions can be employed to remove nutrients from waterways, but feasibility of technology deployment is dependent on the economic viability. Applications of sustainability sciences allows researchers to identify potential solutions to reduce HAB events which are both effective and economically viable. Results show that on average, Lake Erie communities lose $142 M (± $29M) year-1 from HAB's without mitigation. Use of attached-algae systems show an average savings of $12-42M per year from HAB mitigation and represent the most promising technology investigated. Attached-algae systems are the only nutrient reduction technology to show net-positive cash flow when compared with traditional nutrient removal systems. This research dissertation outlines tasks associated with the different applications of sustainability sciences. First, sustainability analyses are used to identify current research roadblocks associated with a technology and are used to identify optimal processing options and provide feedback to researchers to improve these metrics. Next, the tool set was adapted to a novel biorefining process and used to evaluate a value-add proposition for a current technology and showcase current limitations of LCA methodology. And finally, they were leveraged to create a framework for evaluating costs and benefits of technology adoption for pro-active mitigation of environmental disruptions.
Open Access
Investigation of the inhibitory effect of Bacillus pumilus on Nannochloropsis salina
(Colorado State University. Libraries, 2017) Ayshoa Al Gabara, Mirna Dheyaa, author; Reardon, Kenneth F., advisor; Argueso, Cris, committee member; Peebles, Christie, committee member
Microalgae have the potential to be a source of a wide range of industrial materials. To provide the biomass for these products, algae are grown in large volumes. Previous research has shown that there are other microbial species living in algal cultivation systems at these scales, but little is known about the interactions among them. Some of the bacteria in algae cultivations have been identified. Some species can inhibit algal growth, while others are growth promoting. In this research, we focused on one algal species, Nannochloropsis salina, and a bacterial species, Bacillus pumilus. In previous research in our laboratory, B. pumilus culture filtrate had inhibitory effects towards N. salina. We are using these species as a model system to understand a mechanism of bacterial inhibition of algae. Specifically, we have investigated the nature of the inhibitory molecule that is produced by B. pumilus and when it is produced. Our results indicate that B. pumilus produces at least one inhibitory molecule that is probably a protein larger than 30 kD. Since the bacteria produce the highest level of the inhibitory molecule in the presence of marine broth medium (MB), we studied the effects II of the components of MB to determine whether one of these induced the production of the inhibitor more than others. B. pumilus was inoculated in artificial sea water medium (ASW) and several components of MB (peptone, yeast extract and glucose). The filtrate of B. pumilus grown in ASW supplemented with peptone or yeast extract had an inhibitory effect on N. salina, but the filtrate of B. pumilus grown in ASW supplemented with glucose had no inhibitory effect towards the algal species. The results showed that the molecule was produced regardless of the presence of the algal species and it was more concentrated at the late stationary phase. Also there was a certain algal phase when N. salina had more resistance to the inhibition of B. pumilus filtrate. The bacterial species showed the ability to grow on the filtrate of N. salina without any other added components. This knowledge about the mechanism by which this bacterial species inhibits an algae species is useful to determine whether other bacteria use the same strategy and to develop an approach to reduce this inhibitory impact.
Embargo
Involvement of CYP72A219 in herbicide-resistant Palmer amaranth and the role of P450 reductase in the mechanism of metabolic resistance
(Colorado State University. Libraries, 2023) Rigon, Carlos A. G., author; Gaines, Todd A., advisor; Dayan, Franck E., advisor; Beffa, Roland, committee member; Peebles, Christie, committee member
Herbicide resistance in weeds poses a major challenge to modern agriculture worldwide, impacting effective weed control strategies. Metabolic resistance stands out as the major and more complex resistance mechanism due to its ability to metabolize a wide range of herbicides within weed species. Metabolic resistance involves herbicide metabolism through three key phases: activation, conjugation, and sequestration. These phases involve the action of important enzymes such as cytochrome P450 monooxygenases, glutathione S-transferases, and ABC transporters. Metabolic resistance mechanisms have gained prominence in the past decade, posing significant challenges to sustainable agriculture and weed management practices. Amaranthus palmeri (Palmer amaranth) one of the most troublesome weeds globally has evolved metabolic resistance to HPPD inhibitor tembotrione. Understanding and addressing the mechanism are crucial for developing effective strategies to combat herbicide resistance and ensure global crop production. In the present study, four upregulated P450 genes were identified in HPPD-resistant Palmer amaranth from Nebraska (NER), a troublesome weed species. Among these genes, CYP72A219_4284 demonstrated the ability to deactivate the herbicide tembotrione in a heterologous system. This gene was also upregulated in metabolic HPPD-resistant Palmer amaranth plants from different fields across the United States, indicating its involvement in conferring herbicide resistance. Our study also investigated the regulation of these resistance genes, including the promoter sequences and transcription factors involved. Additionally, quantitative trait loci associated with herbicide resistance were identified. This work represents the first identification and validation of genes responsible for herbicide metabolism in Palmer amaranth. Validation of the metabolic resistant gene and the exploration of regulatory mechanisms contribute to a better understanding of metabolic herbicide resistance in weeds, facilitating the development of effective weed management strategies. Cytochrome P450 reductase (CPR), an essential enzyme localized in the endoplasmic reticulum, provides electrons for P450 enzymes during monooxygenase reactions. The transfer of electrons from NADPH to the P450 active site occurs through a complex CPR:P450 interaction. Despite the numerous P450 genes in plant genomes, CPR genes are limited, typically consisting of two or three copies. In Arabidopsis, the two CPR genes, ATR1 and ATR2, have distinct roles in primary and inducible metabolism, respectively. Our study investigated the function of ATR1 and ATR2 in transgenic Arabidopsis plants overexpressing the CYP81A12, which is known to metabolize a wide range of herbicides. The hypothesis was that silencing these ATR1 or ATR2 genes would lead to a reduction of P450 activity involved in herbicide metabolism. ATR1 predominantly transfers electrons to CYP81A12, as knocking down ATR1 led to a significant reduction in herbicide resistance. Knockouts of the ATR2 gene also resulted in decreased herbicide resistance, although the effect was less pronounced. Variation in the number and function of CPR genes among different weed species suggests diverse genetic pressures and potential targets for herbicide resistance management. Inhibition of CPR activity could be a promising approach to restore herbicide effectiveness against metabolic herbicide-resistant weeds. This is the first study to our knowledge that explores the involvement of CPR genes in herbicide resistance in weeds, providing valuable insights into their crucial role. The findings significantly advance our understanding of the mechanisms underlying CPR-mediated herbicide resistance and offer potential targets for the development of effective weed management strategies.
Open Access
Nitrogen utilization in heterotrophic Chlamydomonas reinhardtii
(Colorado State University. Libraries, 2017) Sweeley, Justin Brye, author; Reardon, Kenneth F., advisor; Peebles, Christie, committee member; Snow, Christopher, committee member; Peers, Graham, committee member
The aim of this dissertation research is to bring better understanding to the process of nitrogen adaptation in heterotrophic Chlamydomonas reinhardtii. Microalgae are a diverse group of aquatic photosynthetic organisms that account for almost 50% of the photosynthetic productivity on Earth. There is immense interest in using the unique ability of microalgae to convert sunlight to triacylglycerides (TAGs) for industrial purposes. However, to date there has been little success in implementing these systems at scale and price parity with non-biological methods. Microalgae can modify their metabolism to adapt to the surrounding environment. Under certain circumstances, including nutrient stress, microalgae divert carbon flow away from biomass production and into TAG accumulation. The most common nutrient stress used to trigger TAG accumulation is nitrogen stress, most often induced by transferring a cell from a nitrogen replete medium to a deficient one. The goals of this research were to understand this process, develop methods to manipulate the stress response, and ultimately, to find a way to decouple lipid production from nutrient depletion entirely. Chapter 1 introduces the concepts and research referenced throughout the dissertation including: a background of the C. reinhardtii species, the cultivation techniques that have been applied to cultivation, the physiology behind nitrogen stress, the mechanism that algae use to incorporate nitrogen into the cell, and finally an introduction to the global nitrogen regulator, PII. Chapters 2 through 4 present research into the nitrogen stress pathway and its modification. Chapter 2 discusses a simple method of cultivation used to bring about new insights into the nitrogen stress response, as well as a proposed technique for increasing cellular lipid production. Through differential nitrogen feeding, significantly different effects on cell growth were observed, demonstrating that the response to nitrogen availability is a continuous effect as opposed to an all or nothing "stress response". Chapter 3 describes experiments in which C. reinhardtii was genetically modified to increase understanding of the nitrogen stress response. A nitrogen regulatory protein, PII, was downregulated via amiRNA. Cultures of a mutant strain with lower levels of PII exhibited slow adaptation to fresh nutrient-replete medium but achieved a higher final cell number, final mass concentration, and total neutral lipid content. Similar results were obtained in cultures shifted to nitrogen-free medium. Chapter 4 employs proteomics to identify differences in the specific protein expression pattern between a functional PII strain and a knock-down mutant. Chapter 5 demonstrates a unique approach to producing an engineered nutrient-limited environment in a continuous stirred tank bioreactor. Chapters 7 and 8 summarize the research findings and offer possible direction for future research. Through this research work, new information was obtained on the effects of PII on the cellular response to nitrogen limitation. By increasing our understanding of this basic mechanism, we have proposed several processing conditions that may be implemented to increase microalgal productivity. Furthermore, the homology between microalgae and terrestrial plants suggests the possibility that the results discussed within could give genetic engineers new targets for creating crops with decreased nitrogen demands and increased nitrogen-stress tolerance traits.
Open Access
Orthogonal pair-directed codon reassignment as a tool for evaluating the factors affecting translation in E. coli
(Colorado State University. Libraries, 2018) Schwark, David, author; Fisk, John, advisor; Ackerson, Chris, committee member; Kennan, Alan, committee member; Peebles, Christie, committee member; Snow, Chris, committee member
Proteins are polymers of amino acids that are essential for life, central to cellular function, and have applications in fields ranging from materials science to biomedicine. Proteins in nature are composed of 20 amino acids with limited variability in size and chemical properties. Expanding the genetic code to contain non-canonical amino acids (ncAAs) that contain functionalities not contained in nature is a powerful strategy for probing and extending the properties of proteins. Current in vivo systems for expanding the genetic code have focused on using an engineered orthogonal aminoacyl-tRNA and aminoacyl tRNA-synthetase pairs (tRNA/aaRS) to direct incorporation of ncAAs at amber stop codons. In order to further expand the genetic code to 22 or more amino acids, additional codons must be targeted for reassignment to ncAAs. The genetic code is degenerate; 18 of the 20 canonical amino acids are encoded by more than one codon. Breaking the degeneracy of the genetic code by orthogonal pair directed sense codon reassignment is one pathway to genetic codes of 22 or more amino acids. However, orthogonal pair directed sense codon reassignment is hampered by a limited understanding of the relative importance of the factors that affect the translation of proteins. Here, we describe the repurposing of two commonly used orthogonal pairs from Methanocaldococcus jannaschii (M. jannaschiiI) and Methanosarcina barkeri (M. barkeri) to measure the in vivo reassignment efficiency of 30 different sense codons to tyrosine in E. coli with a simple fluorescence-based screen. The suite of sense codon reassignment efficiencies identified multiple promising codons for reassignment to ncAAs that have not been previously identified. Importantly, every sense codon was partially reassigned to tyrosine when either orthogonal tRNA/aaRS pair was used. Sense codons reassigned to tyrosine with high efficiency may be used directly for reassignment to ncAAs, and any sense codon with measurable reassignment to tyrosine may be improved through directed evolution. The sets of in vivo sense codon reassignment also revealed that E. coli are broadly tolerable to a large number of amino acid substitutions to tyrosine throughout the proteome. The codon reassignment efficiency measurements also enabled an analysis of the in vivo importance of local codon context effects, tRNA abundance, aminoacylation level, tRNA modifications, and codon-anticodon binding energy in determining translational fidelity. Quantitative sense codon reassignment efficiency measurements showed that the process of translation is highly balanced and both tRNA abundance and aminoacylation efficiency do not appear to be dominant factors in determining translational fidelity. Furthermore, quantitative measurements of amber stop codon reassignment efficiencies to tyrosine with the orthogonal M. jannaschii pair revealed that local codon context is an important factor for orthogonal pair directed amber stop codon reassignment.
Open Access
Photoprotection and chloroplast regulation in the green algae Chlamydomonas reinhardtii
(Colorado State University. Libraries, 2019) Cantrell, Michael, author; Peers, Graham, advisor; Pilon, Marinus, committee member; Reddy, A. S. N., committee member; Peebles, Christie, committee member
Absorbed light energy in excess of a cell's photosynthetic capacity can lead to production of reactive oxygen species (ROS) causing cell damage and death. Plants and algae have evolved conserved photoprotective responses that, at the level of light harvesting, are collectively measured as non-photochemical quenching (NPQ) of chlorophyll fluorescence. The major components of NPQ are thermal dissipation of excess light energy (excitation dependent quenching, qE), the migration of antenna complexes from PSII to PSI (state transitions, qT) and inactivation of PSII by damage (photoinhibition, qI). Excess reductant generated during light harvesting can also be dissipated by auxiliary electron transport (AET). The following dissertation aimed to characterize the role of qE in acclimation to saturating and sinusoidal light regimes in the model green algae Chlamydomonas reinhardtii, to characterize potential energy dissipating mechanisms that may occur in absence of qE and identify factors regulating the expression of the chloroplast encoded photosystem I subunit, psaA, using a forward genetic screen. In chapter 2 I show that the qE mutant, npq4lhcsr1, displays decreased growth under a sinusoidal light regime mimicking natural oscillations in irradiance. This reduction in growth rate occurs without a significant impact on carbon accumulation, accumulation of oxidized lipids or impairment of photosynthetic rate. We hypothesized that this was due to increased consumption of excess energy by AET pathways and the results of this investigation are presented in chapter 3. We found that absence of qE in Chlamydomonas did not significantly impact AET associated with light dependent oxygen consumption. The npq4lhcsr1 mutant instead appears to experience less acceptor side limitation downstream of Photosystem I and have a greater capacity for state transitions. This in the absence of any evidence for increased light dependent oxygen consumption in the npq4lhcsr1 mutant indicates that Chlamydomonas compensate for the absence of qE by increasing cyclic electron transport around Photosystem I, which generates additional ATP at the cost of NADPH. In my final chapter I use a positive selectable marker to generate a library of 400 putative psaA mutants, present preliminary flanking sequence characterization for 29 of these mutants and discuss possible roles they may be playing in psaA regulation. Together these chapters expand our understating of the role of qE in long term acclimation to saturating and sinusoidal light regimes and provide a library of putative chloroplast regulatory mutants that, with further characterization, will refine our understanding of chloroplast genome regulation in green algae.
Open Access
Photosynthesis in dynamic and rapidly changing light: the physiology of a cyanobacterium in a photobioreactor
(Colorado State University. Libraries, 2017) Andersson, Bjoern, author; Peers, Graham, advisor; Pilon, Marinus, committee member; Peebles, Christie, committee member
Mass cultivation of aquatic phototrophs in photobioreactors (PBRs) has the potential to produce sustainable biofuels thus reducing net carbon emission and associated climate change. In order to make PBRs productive enough to be economically viable, the biomass accumulation rate and cell density at harvest needs to be high. However, early productivity estimates based on controlled laboratory experiments has not scaled-up to industrial size PBRs. One major reason is that the growth rates in high density, low maintenance PBRs is severely reduced compared to laboratory conditions. This is likely a consequence of the fluctuating light environment. The photophysiological response of algae or cyanobacteria to growth in outdoor PBRs has not been well characterized. The work presented in this thesis aimed to describe the complexity of the light environment in a small-scale PBR and also the physiological response of photoautotrophs to growth in this environment. A dense culture of the cyanobacterium Synechocystis sp. PCC 6803 was grown in a bench-top PBR with an incident light that followed a sinusoidal function peaking at 2000 µmol photons m-2 s-1. These conditions approximate natural sunlight. The diurnal changes in the light environment of the bench top PBR was quantified from the perspective of a single-cell, using a computational fluid dynamic approach (Chapter 1). Due to self-shading within the dense culture, single cells experienced rapid fluctuations (~6 s) between 2000 and <1 µmol photons m-2 s-1, and on average the integrated irradiance per cell was 85% lower than the incident irradiance (mean per cell: 184 µmol photons m-2 s-1). We investigated the activity of photoprotective mechanisms under our realistic light environment, using pulse amplitude modulated (PAM) fluorometery and membrane inlet mass spectrometry (MIMS). Contrary to common assumption we found no evidence for net-photodamage or non-photochemical quenching (NPQ) activity in situ (Chapter 1). In an ex situ experiment we found that alternative electron transport (AET) dissipated 50% of electrons from photosystem II, preventing them from being used for carbon fixation. This indicates that AET, and not NPQ is the first photoprotective mechanism Synechocystis uses under dynamic and fluctuating light. These results have important applications for genetic and metabolic engineering strategies that commonly targets NPQ and photodamage as a way to boost productivity of PBRs. Since, AET caused the main diversion from linear electron transport and carbon fixation, this mechanism should be investigated as a genetic engineering strategy. Samples were also taken to monitor the response of the transcriptome with high temporal precision around the day/night transitions (Chapter 2). The transcriptome data showed that 74% of all genes exhibited some modification in transcription across the diel cycle. In my preliminary analysis of the data (Chapter 2), I found that the major components of photosynthetic light harvesting and electron transport complexes increased in abundance during the whole light period. This is commonly observed in cultures growth under sub-saturating light intensities but not high light stress. Furthermore, few other high light stress responses were observed in the transcriptome. There was little diel variation in transcriptional activity of molecular chaperones (dnaK, hsp, groE families), proteases (ftsH and Deg families), high light inducible proteins (hli), and reactive oxygen species scavengers (superoxide dismutase and catalase peroxidase) that are responsive to high light stress. The flavodiiron proteins are considered the main player of AET in cyanobacteria and are up-regulated transcriptionally under light and inorganic carbon stress. Interestingly, there were no increased abundance in transcripts of the flavodiiron proteins during the light period in my experiment. Assuming that transcript abundance correlates with protein abundance this could mean that either these genes are constituently expressed or that other enzymes may exist that are responsible for the AET. Further analysis of the transcriptomic data and future proteomic analysis may uncover putative genes whose transcriptional pattern indicates that they may play a role in AET under fluctuating light.
Open Access
Physiological response of the Cyanobacterium synechocystis sp. PCC 6803 to fluctuating light
(Colorado State University. Libraries, 2015) Youngblood, Matthew Thomas, author; Peers, Graham, advisor; Peebles, Christie, committee member; Pilon, Marinus, committee member
Photosynthetic microbes are a promising feedstock for renewable biofuels, but the yields of industrial cultivation systems will need significant improvements if they are to be economically viable and succeed. One particular challenge faced by photosynthetic microbes in commercial production systems is the highly dynamic light environment created by vertical mixing within dense cultures. Rapid changes in light intensity make it difficult for these microbes to acclimate and utilize the available light efficiently. Attempts to identify targets for genetically improving photosynthetic microbes to flourish in these environments are hampered by a poor understanding of the physiological response to fluctuating light. My thesis is focused on developing our fundamental understanding of the photophysiology and acclimation responses associated with light environments in industrial conditions. The aim is to eventually increase areal productivity in industrial cultivation systems by applying insights from physiological characterization into future strain engineering approaches. The first chapter introduces the issues associated with industrial cultivation of photosynthetic microbes. I present some background on the need for biofuels, why there has been a focus on using photosynthetic microbes as a feedstock, and how photosynthetic microbes are cultivated industrially. I then present some of the physiological challenges faced by photosynthetic organisms in industrial cultivation, with particular focus on the challenge of a dynamic light environments. I give a brief background on photosynthesis to explain some of the acclimation responses that can be altered in a fluctuating light environment. Chapter 2 presents a proteomic and physiological comparison of the cyanobacterium Synechocystis sp. PCC6803 cultivated in a fluctuating light environment (30s light on/off) to a continuous light environment which had the same average photon flux density. We found that cultures in fluctuating light grew at half the exponential growth rate of continuous light cultures. Reduced growth did not appear to be due to photo-oxidative stress, as we detected reduced levels of reactive oxygen species and oxidative-stress responsive proteins in fluctuating light. We show evidence that reduced growth could be due to a partial shift to a respiratory state in fluctuating light. Reduced growth could also be due to increased dissipation of electrons, as suggested by the higher capacity for photosynthesis at light levels from 106-174 μmol photon m⁻² s⁻¹. We found other surprising changes such as increases in some carbon concentration mechanism components and decreases in some others. These components were thought to be regulated by a similar mechanism due to their co-expression in high CO₂ to ambient CO₂ shift experiments. This suggesting some unusual signaling is occurring due to the fluctuating light. We also found a number of hypothetical and poorly characterized proteins were significantly different in fluctuating and continuous light. In the appendix, I present the preliminary characterization of slr1719, a poorly characterized protein identified in the proteomic analysis of fluctuating and continuous light. This study generated a knock out of slr1719. We found that the Δslr1719 strain compared to a control strain grew much slower in low CO₂ conditions in saturating and sub saturating light conditions. However in replete CO₂ conditions, there was no difference in growth rate, regardless of light intensity tested in Δslr1719 versus the control. We argue that this finding, paired with evidence from the literature, suggests slr1719 may participate in cyclic electron flow. Other roles suggested by the literature for this protein could have important roles in the acclimation to fluctuating light, and warrant further study.