Browsing by Author "Peers, Graham, committee member"

Now showing 1 - 18 of 18

Open Access
A dynamic engineering model of algal cultivation systems
(Colorado State University. Libraries, 2017) Compton, Samuel Lighthall, author; Quinn, Jason C., advisor; Marchese, Anthony, committee member; Peers, Graham, committee member
Proper assessment of the sustainability of algal products is constrained by the onerous process of pilot-scale experimental study. This study developed a bulk growth model that utilizes strain characterization, geospatial data, and cultivation platform geometry to predict productivity across different outdoor systems. The model interprets a minimum of measureable algal strain characteristics along with characteristics of the growth architecture to calculate a time-resolved algal concentration. Validation of the model illustrates an average accuracy of 7.33%+/5.65% for photobioreactors (PBR) and 6.7%+/5.33% for an open raceway pond (ORP) across five total species: Chlorella vulgaris, Desmodesmus intermedius, Galdieria sulphuraria, Galdieria sulphuraria Soos, and Nannochloropsis oceanica. The validated model assesses productivity at several locations in the United States with Chlorella vulgaris, grown in open raceway ponds and Galdieria sulphuraria grown in vertical flat panel photobioreactors. The model investigates seasonal variability through geospatially and temporally resolved extrapolation.
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
Development of a continuous flow ultrasonic harvesting system for microalgae
(Colorado State University. Libraries, 2014) Hincapié Gómez, Esteban, author; Marchese, Anthony J., advisor; Willson, Bryan D., committee member; Dasi, Lakshmi Prasad, committee member; Peers, Graham, committee member
Microalgae have vast potential as a sustainable source of biofuel. However, numerous technoeconomic analyses have indicated that microalgae harvesting represents a critical bottleneck in the microalgae value chain in terms of energy requirements, capital cost and operating cost. This dissertation presents an approach that uses a combination of acoustophoretic, fluid mechanical, and gravitational forces toward the development of a continuous flow microalgae harvesting system. Ultrasonic Standing Waves have been widely reported in the literature as an approach to manipulate particles in a fluid, a phenomena known as acoustophoresis. These waves exert an acoustic force that agglomerate the cells in the wave nodes or antinodes and the force is directly proportional to the cell acoustic contrast factor. Ultrasonic microalgae harvesting is a promising low cost and low energy approach. However, a better understanding of the acoustic properties of microalgae is essential for the development of this technology. Accordingly, a major component of this work focused on accurately quantifying the acoustic contrast factor of microalgae cells of Nannochloropsis oculata, Nannochloropsis gaditana, Phaeodactylum tricornutum and Chlamydomonas reinhardtii by measuring the average cell density and speed of sound using a vibrating tube densitometer. The results indicate a linear correlation of density and speed of sound as a function of cell concentration. Using this correlation, non-scattering volume average relationships were used to compute density and speed of sound for the average algal cell. The acoustic contrast factor was estimated to be between 0.04 - 0.06 for microalgae cells in their corresponding growth media. Second, particle tracking velocimetry was used to determine the magnitude of the acoustophoretic force. In these studies, in addition to microalgae cells, polyamide seeding particles were used as a surrogate. The results obtained conclude that the maximum acoustophoretic forces are approximately 5 pN for Chlamydomonas reinhardtii cells and the results also show that there is change in the acoustic contrast factor from positive to negative with lipid accumulation. This dissertation also presents a novel device for the acoustic harvesting of microalgae. The design is based on using the acoustophoretic force, acoustic transparent materials and inclined settling (Boycott effect). A filtration efficiency of 70% ± 5% and a concentration factor of 11.6 ± 2.2 were achieved at a flow rate of 25 mL • min-1 and an energy consumption of 3.6 ± 0.9 kWh • m-3. The effects of the applied power, flow rate, inlet cell concentration and inclination were explored. It was found that the filtration efficiency of the device is proportional to the power applied. However, the filtration efficiency experienced a plateau at a 100 W • L-1 of power density applied. The filtration efficiency also increased with increasing inlet cell concentration and was inversely proportional to the throughput of the device as measured flow rate. It was also found that the optimum settling angle for maximum concentration factor occurred at an angle of 50° ± 5°. At these optimum conditions, the device had higher filtration efficiency in comparison to other similar devices reported in the previous literature.
Open Access
Development of an organically certifiable growth medium for N-fixing cyanobacteria in a raceway biofertilizer production system
(Colorado State University. Libraries, 2014) Barminski, Rosalyn, author; Davis, Jessica, advisor; Storteboom, Heather, committee member; Peers, Graham, committee member
The on-farm cultivation of N-fixing cyanobacteria in raceway ponds may provide an alternative N source in organic farming systems. The cultivation of cyanobacteria in an organic farming system requires an organically certifiable growth medium. Additionally, efficient cyanobacterial cultivation depends on production methods that reduce the severity of the three growth limiting factors present in outdoor raceway cultivation: inefficient solar irradiance, growth medium nutrient depletion, and day-night temperature fluctuations. The purpose of this work was two-fold, first to develop and test an organically certifiable growth medium, and secondly to test four specific production methods so as to optimize cyanobacterial growth and N-fixation. The four raceway production methods tested separately included: batch (B) versus semi-continuous (SC) operation mode, a culture depth of 20-cm versus 25-cm, bicarbonate supplementation in the growth medium, and four different cover plastics over raceways. All studies used a cyanobacterium cultured from a Fort Collins, CO lake, with 99% similarity to Anabaena cylindrica. Cyanobacterial growth was estimated by optical density (OD) and chlorophyll content and cyanobacterial N-fixation was estimated by net Total Kjeldahl Nitrogen (TKN). In chapter 2, "Comparison of cyanobacterial growth and nitrogen fixation in a newly developed organically certifiable growth medium and Allen and Arnon growth medium", a laboratory and raceway study were conducted. In the lab study, the nutrients of Allen and Arnon (AA) that were not organically certifiable were replaced with organically certifiable nutrients to compose the organic medium (RB). The exponential growth rate was significantly higher in the RB medium compared to AA. Conversely the net TKN in the RB medium was 37% lower than that of AA. The lower N-fixation in the RB medium was attributed to the presence of N in the P source used for RB medium (bone meal). In the raceway study, there was no significant difference in growth between the two treatments despite lower concentrations of P, Co, Zn, and B in the RB medium. An overarching limiting factor evident in both treatments such as light limitation or C depletion could explain why there was no observed growth effect due to the low P, Co, Zn, and B concentrations of RB medium. The net TKN between the two treatments was not statistically different, which suggests similar N-fixation. The conclusion of similar N-fixation was questioned due to the contribution of dissolved N from bone meal. Together, the studies support that the RB medium supports growth similar to that of the AA medium in raceway cultivation. However, since N was present in the RB medium, it is possible that maximal N- fixation was not achieved. Recommendations to increase nutrient concentrations in RB medium are discussed in chapter 4, "Future recommendations". In chapter 3, "Biomass yield and nitrogen fixation of cyanobacteria in outdoor raceways under batch versus semi-continuous operation", a SC treatment was operated under a 25% harvest regime every other day beginning on day 6. The B treatment was grown for 14 days, then 85% of the treatment was harvested and the remaining 15% was used as seed to begin a second B set. At the end of four weeks, biomass yield and total N fixed was calculated for the B and SC treatments. There was no difference in biomass yield or N yield between the two treatments. More than likely the SC was harvested when the culture density was above the optimal cell density range, resulting in a lower total biomass and N yield than what could have been achieved within the optimal cell density range. Determination of the optimal cell density and a specific harvest regime that maintains the SC within the optimal cell density would result in a higher total SC biomass and N yield compared to that of B. Possible experiments to determine the optimal cell density are discussed in chapter 4, "Future recommendations". In Appendix II, "Cyanobacterial growth and nitrogen fixation in response to depth, bicarbonate supply, and hoop house coverings in outdoor culture", three separate batch studies were conducted in 1.2-m (l) by 0.6-m (w) by 0.3-m (h) tanks. The first experiment compared the growth and N-fixation of batch cultures grown at two different depths (20-cm and 25-cm). Raceway depth did not have an effect on total growth or net N-fixation. The second experiment compared cyanobacterial growth and N-fixation in AA medium supplied with 0 mM (control), 0.2 mM (low treatment), and 2.0 mM (high treatment) of potassium bicarbonate (KHCO3). There was no increase in growth or N-fixation due to addition of KHCO3. It was concluded that inadequate KHCO3 was added to significantly increase growth and that the addition of NaHCO3 rather than KHCO3 is necessary to assure adequate Na concentrations needed for maximal bicarbonate uptake. The third experiment compared the growth and N-fixation of cultures grown under different hoop house plastics (Thermax, Luminance, Dura-film Super 4, and 4 mil Husky construction plastic) and a no-cover control. None of the covers tested in the study increased the growth compared to the no-cover control. Zn slowly leached from the cultivation tanks, so that by the end of the third study, Zn toxicity clouded the interpretation of results.
Open Access
Development of genetic parts for improved control of translation initiation in Synechocystis sp. PCC 6803 with an application in biofuel production
(Colorado State University. Libraries, 2021) Sebesta, Jacob, author; Peebles, Christie A. M., advisor; Peers, Graham, committee member; Prasad, Ashok, committee member; Reardon, Kenneth, committee member
Metabolic engineering is developing into a field that can change the way we produce a wide variety of valuable chemicals. Many chemicals are already produced in microbial cultures. Metabolic engineering enables us to modify organisms to produce metabolites they don't usually produce, assuming an enzyme can be identified in another organism that catalyzes the formation of that product (or an enzyme can be designed for that task through protein engineering). The distribution of accumulated metabolites can also be altered. There are some cases where metabolites can be accumulated through cultivation practices. Methods of metabolic engineering to overexpress, knockdown, or knockout native enzymes provide additional tools to alter cellular metabolism and drive accumulation of those products. Precise control over gene expression is central to these efforts. To avoid competition with human food crops and the resources need to produce them, cyanobacteria may be utilized for production of valuable chemicals. Through photosynthesis, they can utilize carbon dioxide from geological formations or from industrial waste streams. Since most metabolic engineering has been developed in E. coli and yeast, it was necessary to first adapt the basic methods for use in cyanobacteria. Along with my co-authors Dr. Allison Werner and Dr. Christie Peebles, we reviewed methods for producing genetically modified Synechocystis Sp. PCC6803 (S. 6803). To facilitate the generation of strains with many modifications, we covered the method developed in the Peebles Lab for making markerless selections which remove any antibiotic selection markers. A previous graduate student in the Peebles lab, Stevan Albers, found that strong promoter-ribosome binding site combinations that drove high expression of GFP did not necessarily result in high expression when used to drive expression of a different gene. Therefore, in our work to produce bisabolene in S. 6803 we tested many ribosome binding sites. In addition, we tested five different codon optimizations of the bisabolene synthase to ensure that expression was not prevented by slow translation elongation. We found that the simple measure of the codon adaptation index (CAI) correlated with expression of the five different codon optimizations. Using a thermodynamic model of translation initiation, we designed ten ribosome binding sites to increase bisabolene synthase expression by 10-fold. Only one of those designs actually approached a 10-fold increase, highlighting the need to continue testing several ribosome binding sites to achieve a desired expression level. Since industrial cultivation of cyanobacteria occurs outdoors, subject to natural light:dark cycles, we tested two of the designed strains in light:dark cycles. The strains reached similar bisabolene titers after being exposed to the same amount of total light period as those previously tested in continuous light. Overall, this work increased the highest bisabolene titer reported in cyanobacteria by approximately 10-fold. The need to test many ribosome binding sites limits progress in cyanobacterial metabolic engineering. The research of others suggest that ribosome binding sites interact with coding sequences by forming secondary structures with different free energy of folding. The estimation of the free energy of folding may be inaccurate, and, further, the kinetics of such folding may also be important to translation initiation rates. We tested two different designs to limit the impacts that secondary structures that span either side of the start codon may have on translation initiation rates in both E. coli and S. 6803. Utilization of a 21-nucleotide leader sequence after the start codon to make the sequence context consistent for ribosome binding sites between different coding sequences did not improve the correlation found between the expression of two different reporter genes in either organism. Bicistronic designs use translational coupling between an upstream open reading frame and the gene of interest with a ribosome binding site contained within the upstream open reading frame to re-initiate translation. This design exploits the helicase activity of ribosomes in elongation mode to actively unfold the secondary structure around the start codon of the gene of interest. We expected this activity to reduce the impacts of secondary structure and improve the correlation in expression between two different reporter genes. Intriguingly, the correlation was much improved in E. coli, but not in S. 6803. Together, this dissertation suggests that there are important differences in translation initiation between E. coli and S. 6803. Improved ribosome binding site design for cyanobacteria would facilitate further increases in terpenoid production both by enabling higher expression of heterologous terpenoid synthases and by reducing the number of strains that must be tested to achieve the desired expression level for each enzyme. Future directions suggested by this work include studies of translation initiation mechanisms in cyanobacteria, development of cell-free expression systems to facilitate rapid testing of many different genetic constructs, and further efforts at pathway engineering to increase terpenoid titer and productivity in cyanobacteria.
Open Access
Genome-scale metabolic modeling of cyanbacteria: network structure, interactions, reconstruction and dynamics
(Colorado State University. Libraries, 2016) Joshi, Chintan Jagdishchandra, author; Prasad, Ashok, advisor; Peebles, Christie A. M., committee member; Reardon, Kenneth, committee member; Peers, Graham, committee member
Metabolic network modeling, a field of systems biology and bioengineering, enhances the quantitative predictive understanding of cellular metabolism and thereby assists in the development of model-guided metabolic engineering strategies. Metabolic models use genome-scale network reconstructions, and combine it with mathematical methods for quantitative prediction. Metabolic system reconstructions, contain information on genes, enzymes, reactions, and metabolites, and are converted into two types of networks: (i) gene-enzyme-reaction, and (ii) reaction-metabolite. The former details the links between the genes that are known to code for metabolic enzymes, and the reaction pathways that the enzymes participate in. The latter details the chemical transformation of metabolites, step by step, into biomass and energy. The latter network is transformed into a system of equations and simulated using different methods. Prominent among these are constraint-based methods, especially Flux Balance Analysis, which utilizes linear programming tools to predict intracellular fluxes of single cells. Over the past 25 years, metabolic network modeling has had a range of applications in the fields of model-driven discovery, prediction of cellular phenotypes, analysis of biological network properties, multi-species interactions, engineering of microbes for product synthesis, and studying evolutionary processes. This thesis is concerned with the development and application of metabolic network modeling to cyanobacteria as well as E. coli. Chapter 1 is a brief survey of the past, present, and future of constraint-based modeling using flux balance analysis in systems biology. It includes discussion of (i) formulation, (ii) assumption, (iii) variety, (iv) availability, and (v) future directions in the field of constraint based modeling. Chapter 2, explores the enzyme-reaction networks of metabolic reconstructions belonging to various organisms; and finds that the distribution of the number of reactions an enzyme participates in, i.e. the enzyme-reaction distribution, is surprisingly similar. The role of this distribution in the robustness of the organism is also explored. Chapter 3, applies flux balance analysis on models of E. coli, Synechocystis sp. PCC6803, and C. reinhardtii to understand epistatic interactions between metabolic genes and pathways. We show that epistatic interactions are dependent on the environmental conditions, i.e. carbon source, carbon/oxygen ratio in E. coli, and light intensity in Synechocystis sp. PCC6803 and C. reinhardtii. Cyanobacteria are photosynthetic organisms and have great potential for metabolic engineering to produce commercially important chemicals such as biofuels, pharmaceuticals, and nutraceuticals. Chapter 4 presents our new genome scale reconstruction of the model cyanobacterium, Synechocystis sp. PCC6803, called iCJ816. This reconstruction was analyzed and compared to experimental studies, and used for predicting the capacity of the organism for (i) carbon dioxide remediation, and (ii) production of intracellular chemical species. Chapter 5 uses our new model iCJ816 for dynamic analysis under diurnal growth simulations. We discuss predictions of different optimization schemes, and present a scheme that qualitatively matches observations.
Open Access
Investigating the potential of meltwater as a local source of ice nucleating particles in the central Arctic summer
(Colorado State University. Libraries, 2024) Mavis, Camille, author; Kreidenweis, Sonia, advisor; Creamean, Jessie, advisor; Pierce, Jeffrey, committee member; Peers, Graham, committee member
Due to climate change, the Arctic has crossed a threshold into positive feedbacks between sea-ice loss and increased absorption of solar radiation, causing warming up to four times the global average. Parameterizing the Arctic radiation budget to predict the new steady-state is paramount for guiding policies impacting future global socioeconomics and Arctic livelihoods. Arctic mixed-phase clouds (AMPCs) are a pillar in the feedback systems by modulating the surface energy budget, depending on the partitioning of cloudwater between ice and liquid phases that is sensitive to the concentration of ice nucleating particles (INPs) in the atmosphere. However, current observational gaps of central Arctic INP concentrations and sources may contribute to current challenges in resolving the controls on Arctic cloud ice content. The year-long expedition aboard the RV Polarstern from 2019 - 2020, entitled The Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC), was a highly coordinated interdisciplinary effort that provided a unique opportunity to observe INPs in the central Arctic. The Arctic summer is a unique period characterized by pristine aerosol conditions, in which emissions from local sources have an increased influence, potentially impacting the ubiquitous low-lying AMPCs. Thus, the summer is an ideal season for exploration of the potential importance of INPs from local sources, such as melt ponds. In this study, we used the Colorado State University (CSU) Ice Spectrometer and chemical treatments to determine the INP concentration and inferred composition in source samples of bulk sea water and meltwater from ponds and leads over the month of July. In addition, ambient aerosol filters were deployed both on the ship and on the ice, downwind of these meltwater features. We found that the concentration of INPs in meltwater was 10 times higher than in the mixed layer of the ocean, a surprising result since previous studies did not see a difference in the two source samples. The INPs in meltwater were capable of freezing at temperatures (T) ≥ −10 °C and were predominantly biological, based on our heating assay. Biological INPs capable of freezing at T ≥ −10 °C were present in 80 % of the on-ice aerosol samples. The alignment of slopes of the cumulative INP spectra between the meltwater and aerosol filter samples at T ≥ −15 °C suggested an influence from meltwater on the aerosol INPs at those temperatures. Similarities between aerosol INP sampled on the ice and on-board Polarstern suggested that the on-ice INP concentrations were likely influenced by a regional meltwater source signature, rather than being measurably impacted by a singular upwind pond. A relationship was observed between wind speed, supermicron particle counts, and on-ice aerosol INP populations active at warm (−15 °C) and cold (−25 °C) temperatures. A distinct on-ice aerosol sample containing no INPs active at T ≥ −15 °C was found to be influenced by southerly air over the ice-free ocean, emphasizing the potential impact meltwater may have as a unique source of warm temperature INPs in the central Arctic. These findings suggest that summertime central Arctic biological INP concentrations may increase if, as predicted, a spatio-temporal expansion of the melt season occurs in the near future. This increased INP concentration from local sources could impact central Arctic cloud microphysics, and thus their impact on the surface energy budget.
Open Access
Iron economy in Arabidopsis thaliana rosettes
(Colorado State University. Libraries, 2014) Hantzis, Laura, author; Pilon, Marinus, advisor; Jahn, Courtney, committee member; Peers, Graham, committee member
Iron is important for plant growth and lack of iron negatively affects crop productivity. When we understand more about how plants prioritize their iron use we can use this knowledge to benefit the people of the world. Plant metabolism can be altered to allocate iron in such a way creating larger and/or healthier edible parts. In the first chapter of this thesis I briefly discuss the biological roles of iron and its function in plants with an emphasis on photosynthesis. In the second chapter I investigated the potential prioritization of iron-dependent proteins in Arabidopsis thaliana plants that were grown hydroponically and exposed to one week of iron deprivation followed by one week of iron resupply. Through the one week of iron depletion the treated plants became visually chlorotic and after one week of iron recovery the treated plants appeared to have fully recovered from the chlorosis. At the end of the recovery week treated plants had significantly less biomass than the control plants yet suffered no indirect effects. To investigate if the decrease in biomass was caused by defects in photosynthetic electron transport chlorophyll fluorescence was measured as well as the photooxidation-reduction values of photosystem I (PSI). Indeed photosynthesis was affected and it was found that there was a decrease in electrons flowing to PSI. With the use of immunoblots it was discovered that the cytochrome b6f complex proteins were strongly affected by iron depletion followed by the subunits of PSI. Furthermore, I found that the abundance of sulfur metabolism proteins decreased in reaction to decreased iron nutrition. In the third chapter I discuss the implications of my findings for plant science and society and I give recommendations for follow up work on this project.
Open Access
Loss and integration: tracing the functional replacement of mitochondrial tRNA genes
(Colorado State University. Libraries, 2021) Warren, Jessica Marie, author; Sloan, Daniel, advisor; Mueller, Rachel, committee member; Reddy, Anireddy S. N., committee member; Peers, Graham, committee member; Stasevich, Timothy, committee member
To view the abstract, please see the full text of the document.
Open Access
Metabolic engineering of cyanobacteria: developing molecular tools and characterizing strain performance in light:dark cycles
(Colorado State University. Libraries, 2015) Cheah, Yi Ern, author; Peebles, Christie M., advisor; Reardon, Kenneth F., committee member; Prasad, Ashok, committee member; Peers, Graham, committee member
The conversion of CO2 and light energy to biofuels holds promise for a renewable and environmentally responsible source of energy that could meet the growing demand for transportation fuels. However, early efforts to commercialize biofuels from plants were hampered by social, economic, and technological difficulties. Photosynthetic microbes present an opportunity for a more efficient conversion of fixed carbon to biofuels by bypassing the need of harvesting sugars from plants to be fermented by heterotrophic bacteria. More recently, cyanobacterial technologies have received considerable interest due to their ease of genetic manipulation that enables them to produce a myriad of biofuels and biochemicals directly from CO2. This relatively nascent technology needs to be developed in order to realize its commercial potential. Metabolic engineering is the systematic improvement of strains through the use of a variety of theoretical and experimental techniques. To date, heterologous pathways expression has been the most successful in model heterotrophic organisms (e.g. E. coli) and advances from these systems have to be carefully transferred over to cyanobacteria. Though several studies have demonstrated the capability of engineering cyanobacteria to produce biofuels, there is yet to be any commercially feasible production platform of fuels from CO2. Amongst the challenges is the need to improve yields and titers from recombinant strains. However, the physiology of cyanobacteria is distinct from that of heterotrophic organisms and therefore requires careful design and study in order to optimize for higher yields. This thesis contributes several technologies to foster the scale-up of cyanobacteria systems from the bench to industrial scale. We first developed a markerless chromosomal modification method in WT Synechocystis PCC6803 that could reduce the metabolic load and cultivation cost compared to plasmid-based expression methods. We established a counter-selection method that necessitates two rounds of modifications in order to screen for the desired mutant harboring the gene(s) of interest. In the first round, a synthetic circuit consisting of a nickel inducible toxin gene (mazF) and a kanamycin resistance marker is integrated into a specific locus in WT Synechocystis. In the second round, a construct harboring gene(s) of interest is transformed into the prerequisite strains and screen on Ni2+ to obtain the desired mutants. Next we established a free fatty acid (FFA) producing platform in Synechocystis PCC6803 by pursuing three goals: 1) deletion of acyl-acyl carrier protein (acyl-ACP) synthetase (aas), 2) optimize the expression of thioesterase I (TesA) with a promoter library and 3) examine the effects of light:dark cycles on FFA production in Synechocystis. For the first goal, we were successful in engineering an aas deletion strain that had increased FFA production. In the second goal, we developed four Synechocystis variants with increasing TesA expression strengths from the aas deletion strain. No increase in FFA production was observed between the TesA expressing strains (with aas deleted) compared to the baseline aas deletion strain. On the protein level, we found no evidence of TesA enzyme activity even though TESA peptides were detected in our Synechocystis strains. In the third goal, we learn that diel light:dark cycles causes a significant decrease in production of FFAs in FFA producing mutants of Synechocystis compared to continuous light. We did not observe any transcriptional changes in the fatty acid biosynthesis pathway between our WT and FFA producing strains to explain these changes. In summary, this thesis is impactful in two ways: 1) it entails the development of a markerless genetic modification method for use in cyanobacteria and 2) it characterizes the production of FFAs from engineered cyanobacteria under diel light:dark cycles. Overall, this thesis helps address the difficulties in the development of cyanobacteria systems for eventual use in an industrial setting.
Open Access
Metabolic engineering of the cyanobacterium Synechocystis sp. PCC 6803 for the production of astaxanthin
(Colorado State University. Libraries, 2016) Albers, Stevan Craig, author; Peebles, Christie A. M., advisor; Reardon, Kenneth, committee member; Prasad, Ashok, committee member; Peers, Graham, committee member
Synechocystis sp. PCC 6803 is a photosynthetic eubacterium capable of using light energy to generate biomass from atmospheric CO2 and is considered to be the model organism of photosynthetic microbes. Much of the knowledge accumulation related to this organism has centered on the cellular photosynthetic process because this organism has many similarities to the chloroplasts of higher order plants. Synechocystis also shows great promise as a microbial cell factory, as scientific studies describing metabolite production from this organism continue to accumulate in the literature. While these studies highlight the considerable amount of gains made in regards to production in Synechocystis, they also shed light on the considerable amount of gaps in knowledge regarding many aspects of this organism. As the field of metabolic engineering continues to grow within Synechocystis, researchers must continue to develop production pathways that leverage comprehensive engineering strategies that help in shedding light on critical engineering hurdles. This information is critical for the successful development of photosynthetic microbes as cellular production platforms capable of generating titers similar to those seen in other cellular systems utilized to generate economically viable metabolites for humankind. In this work, we utilized several metabolic engineering strategies to manipulate the carotenoid biosynthesis pathway in Synechocystis for the production of the non-native carotenoids, astaxanthin as well as canthaxanthin. A Synechocystis mutant was engineered with an insertion of a β-carotene di-ketolase gene crtW148 from Nostoc punctiforme, insertion of an additional copy of the endogenous β-carotene hydroxylase gene crtR from Synechocystis, and an open reading frame disruption of the endogenous β-carotene mono-ketolase gene crtO. These manipulations generated a mutant capable of an increase in the overall carotenoid content by 178 ± 10% % of that seen in wild type cells as well as astaxanthin titers that reached production rates of 1.11 ± 0.07 mg/l/day and canthaxanthin titers reaching 1.49 ± 0.05 mg/l/day. To add upon this work, we leveraged several promoters, the PSCA6-2 promoter as well as the PsigA promoter to control the expression of the crtW148 gene within several constructs. These promoters were generated in a research study we performed that leveraged rational design strategies to develop a suite of promoters capable of driving gene expression as various strengths within Synechocystis. This study generated a library of 10 promoter-constructs capable of a dynamic range of expression strength, exhibiting a 78 fold change between the lowest expressing promoter, Psca8-2 and the highest expressing promoter, Psca3-2 when tested within Synechocystis. Use of the PSCA6-2 promoter within the carotenoid pathway engineering experiment increased carotenoid production of target carotenoids by 150% to 197% over production seen from the same constructs run by the promoter PsigA. In addition to engineering of the carotenoid biosynthesis pathway, we also tested the impacts of diel cycle light conditions on carotenoid production and accumulation. When exposed to 12 hour light/dark conditions, the mutant crtR::cruB::ΔcrtO-PSCA6-2::crtW generates carotenoids at rates of 43 ± 14.8 % of that of the same culture grown in constant light conditions. We hypothesized that this lag was caused by the endogenous cellular control of the carotenoid pathway initiated by the metabolic burden placed on the cell. We also hypothesize that this metabolic burden was caused by the engineered constitutive expression of the astaxanthin producing genes during dark conditions. To address potential concerns of constitutive expression of pathway genes during stress conditions like the dark conditions highlighted in the astaxanthin work, our lab constructed a chemically inducible construct for use in Synechocystis that is based on the tac repressor. Upon chemical induction with IPTG, this same mutant strain was capable of exhibiting an average 24X increase in GFP expression over that of the repressed state. In addition to this work, we studied several light induced promoters to better understand their ability to control gene expression during various light conditions in neutral locations within the Synechocystis genome. We identified that the PpsbAII promoter functions very differently in light and dark conditions when it is moved from its native location within the genome. As many researchers utilize this promoter to control gene expression, this information may be critical to fully understanding gene expression of pathways leveraging this promoter construct. Three additional promoter constructs, the PpsbAIII. PgroEL2, and PsigD promoters were also tested for differential expression in light and dark conditions within the neutral region slr0168. Additionally, nucleotide mutations were made to regions within the PpsbAII promoter, to better understand this promoter’s sensitivity to varying light intensities.
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
Parasites of two closely related Poeciliid species across a salinity gradient on the island of Trinidad: implications for geographic range limits
(Colorado State University. Libraries, 2018) Robison, Porsche, author; Ghalambor, Cameron, advisor; McGrew, Ashley K., committee member; Schaffer, Paula, committee member; Peers, Graham, committee member
Parasite communities can vary greatly both within and amongst host populations. Many factors may be responsible for this variation in parasite diversity, and parasite-host relationships are of great ecological importance as parasites can alter host behavior, impact population demography, and drive co-evolutionary dynamics. One long-standing ecological question is how these parasite-host interactions shape mutual geographic distributions, which are also impacted by various abiotic and biotic factors. However, few studies have investigated how parasite communities change across environmental gradients and different host species, or how parasite abundance changes within and outside the host geographic range. The island of Trinidad provides a model system that can be used to address these questions. On this island, the tropical fish Poecilia reticulata inhabits mountainous and lowland freshwater streams but avoids brackish waters. A close relative, Poecilia picta inhabits both lowland freshwater and brackish water streams. To date, no study has investigated how internal parasite communities vary across this salinity gradient or between these two closely related host species with overlapping geographic ranges. In lab studies, P. reticulata has been shown to be physiologically tolerant of brackish water, suggesting some other environmental factor may limit their dispersal and range expansion into brackish waters. Here we investigated how internal parasite diversity changes between 1) natural P. reticulata and P. picta populations in freshwater, 2) natural populations of P. picta in fresh and brackish water sites, and3) P. reticulata, experimentally exposed to fresh and brackish water conditions. We found the prevalence of digenean trematode metacercariae to be 100% across three different river systems for both host species, however, mean metacercarial abundance differed significantly by river. Based on morphological differences in the metacercariae, we identified three distinct morphospecies. All three morphospecies were found in freshwater P. reticulata and P. picta. However, mean abundance of parasites varied across the two host species with P. reticulata harboring more parasites, on average, compared to P. picta. All three morphospecies were also found in P. picta in brackish water, but the total mean metacercarial abundance of P. picta was found to be increased in brackish compared to freshwater sites. Collectively, these results suggest that the three morphospecies utilize both hosts and they are limited in their geographic distribution by salinity. Still, the internal abundance of parasites varies between the two hosts depending on the salinity. We tested whether these same parasites may be limiting the distribution of P. reticulata to freshwater, by experimentally exposing P. reticulata populations to field-collected brackish water. We assumed field-collected brackish water contains live cercaria and conducted exposures for a period of seven days. Compared to controls exposed to field-collected freshwater, there was a significant increase in the internal metacercarial abundance, along with an increase in mortality amongst the brackish water exposed group. These results suggest that movement of P. reticulata into novel brackish environments may be inhibited by increased parasitism; however, further investigations are warranted to better understand the mechanisms that determine the geographic distributions of parasites and their hosts.
Open Access
Selenium uptake, differentiation and metabolism in hyperaccumulator Stanleya pinnata
(Colorado State University. Libraries, 2013) Harris, Jonathan, author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Peers, Graham, committee member; Ward, Sarah, committee member
Selenium (Se) is a biologically essential element for many animals, some prokaryotes and algae. However, even in organisms that require Se, the range between sufficiency and toxicity for Se is narrow. Although there are no reports of a Se requirement or selenoproteins in higher plants, there are species that appear endemic to seleniferous soil and concentrate Se in their leaves to levels exceeding 1000 mg kg-1 dry weight. These plants are known as Se hyperaccumulators and have an exceptional ability to tolerate and enrich themselves with this toxic element. As a result of the Se concentrations in their tissues, Se hyperaccumulators are extremely toxic to most organisms. Studies have found that Se hyperaccumulation protects these plants from many herbivores and pathogens as an "elemental defense." Some of these hyperaccumulators have been studied for their use in phytoremediation of naturally occurring and anthropogenically contaminated seleniferous soils. Although the slow growth of most hyperaccumulators limits their direct application for phytoremediation, they can be utilized as a source of genes to genetically enhance Se accumulation and tolerance in popular phytoremediator species. The goal of this study is to better characterize the uptake, metabolic fate and molecular mechanisms responsible for Se tolerance in Stanleya pinnata, a hyperaccumulator in the Brassicacae. Two main techniques were utilized: physiological experiments followed by elemental analysis to characterize Se uptake and interactions with the related element sulfur (S), and Illumina sequencing of the transcriptomes of Stanleya pinnata and related non-hyperaccumulator Stanleya elata. The first chapter presents a literature review of Se hyperaccumulation: what is known about Se assimilation in higher plants, and some unique characteristics of hyperaccumulators. The metabolism of Se through the sulfate assimilation pathway is described, and known mechanisms of Se tolerance and accumulation in representative plants are reviewed. In addition, some of the previous work on Stanleya is reviewed, including a number of studies that have shown ecological benefits of Se hyperaccumulation. Known beneficial genes for Se tolerance and accumulation are discussed in the context of phytoremediation. In chapter 2, Se-specific uptake was tested in two ecotypes of S. pinnata, and contrasted with related non-hyperaccumulator Brassica juncea. To test for Se specificity of sulfate transporters, plants were supplied with varying concentrations of selenate and two concentrations of sulfate. The results showed that S. pinnata is able to take up large amounts of Se, even at exceedingly low supplied Se:S ratios. In addition, S. pinnata preferentially mobilized large amounts of Se to young leaves, without commensurate mobilization of S. These trends were not observed in the non-hyperaccumulator B. juncea, which showed dramatically reduced Se uptake under elevated sulfate supply. Moreover, there was no evidence of preferential allocation of Se to young tissues in B. juncea. Taken together, these findings support the hypothesis that Stanleya contains transporters with an increased specificity for Se, allowing it to take up preferentially and mobilize Se over S. Since previous work has shown that molybdate may be taken up in part by plant sulfate transporters, this element was also monitored. It was observed that increasing supply of selenate and sulfate significantly reduced the molybdenum (Mo) content of leaves in S. pinnata. In contrast, B. juncea showed an increase in Mo content with increases in supplied selenate. In the experiment described in Chapter 3, Illumina sequencing was performed to compare the root and shoot transcriptomes of hyperaccumulator S. pinnata and non-hyperaccumulator S. elata in the presence or absence of selenate. An overview is presented of the overall transcriptome response patterns, followed by a more detailed analysis of transcripts involved in S/Se metabolism. In the presence of Se, 40 of the 56 S/Se-related genes were more highly expressed in S. pinnata than S. elata. Particularly promising findings include a vastly upregulated root sulfate/selenate transporter (Sultr1;2) and ATP sulfurylase (APS2). Lastly, some preliminary findings are presented from several biochemical approaches used to further investigate S. pinnata hyperaccumulation mechanisms. Organic forms of Se were investigated in S. pinnata and S. elata using a newly developed liquid chromatography mass spectrometry (LC-MS) method. It was shown that S. pinnata accumulates significant amounts of selenocystathionine as well as methyl-selenocysteine. Moreover, activities of selenocysteine lyase (SL) and cysteine desulfurase (CysD) were investigated in S. pinnata and S. elata, which revealed strong SL activity in the hyperaccumulator. The possible role of this enzyme in Se hyperaccumulation remains to be elucidated. Finally, superoxide dismutase activities were compared between the two species in relation to Se supply. Stanleya pinnata and other Se hyperaccumulators may be valuable resources for genes involved in Se tolerance and hyperaccumulation, to create genetically engineered plants for phytoremediation purposes. In addition to the potential environmental benefits, understanding potential biological roles for Se and its metabolism in these plants may have broad applications for human health. Many organic seleno-compounds have been studied for their anti-carcinogenic properties in multiple systems and types of cancer. Efficacy of these Se compounds appears to vary based on the form of Se. Plants capable of creating different forms of organic Se may become a valuable pharmaceutical resource.
Open Access
Statistical modeling and inference for complex-structured count data with applications in genomics and social science
(Colorado State University. Libraries, 2020) Cao, Meng, author; Zhou, Wen, advisor; Breidt, F. Jay, advisor; Estep, Don, committee member; Meyer, Mary C., committee member; Peers, Graham, committee member
This dissertation describes models, estimation methods, and testing procedures for count data that build upon classic generalized linear models, including Gaussian, Poisson, and negative binomial regression. The methodological extensions proposed in this dissertation are motivated by complex structures for count data arising in three important classes of scientific problems, from both genomics and sociological contexts. Complexities include large scale, temporal dependence, zero-inflation and other mixture features, and group structure. The first class of problems involves count data that are collected from longitudinal RNA sequencing (RNA-seq) experiments, where the data consist of tens of thousands of short time series of counts, with replicate time series under treatment and under control. In order to determine if the time course differs between treatment and control, we consider two questions: 1) whether the treatment affects the geometric attributes of the temporal profiles and 2) whether any treatment effect varies over time. To answer the first question, we determine whether there has been a fundamental change in shape by modeling the transformed count data for genes at each time point using a Gaussian distribution, with the mean temporal profile generated by spline models, and introduce a measurement that quantifies the average minimum squared distance between the locations of peaks (or valleys) of each gene's temporal profile across experimental conditions. We then develop a testing framework based on a permutation procedure. Via simulation studies, we show that the proposed test achieves good power while controlling the false discovery rate. We also apply the test to data collected from a light physiology experiment on maize. To answer the second question, we model the time series of counts for each gene by a Gaussian-Negative Binomial model and introduce a new testing procedure that enjoys the optimality property of maximum average power. The test allows not only identification of traditional differentially expressed genes but also testing of a variety of composite hypotheses of biological interest. We establish the identifiability of the proposed model, implement the proposed method via efficient algorithms, and expose its good performance via simulation studies. The procedure reveals interesting biological insights when applied to data from an experiment that examines the effect of varying light environments on the fundamental physiology of a marine diatom. The second class of problems involves analyzing group-structured sRNA data that consist of independent replicates of counts for each sRNA across experimental conditions. Most existing methods—for both normalization and differential expression—are designed for non-group structured data. These methods may fail to provide correct normalization factors or fail to control FDR. They may lack power and may not be able to make inference on group effects. To address these challenges simultaneously, we introduce an inferential procedure using a group-based negative binomial model and a bootstrap testing method. This procedure not only provides a group-based normalization factor, but also enables group-based differential expression analysis. Our method shows good performance in both simulation studies and analysis of experimental data on roundworm. The last class of problems is motivated by the study of sensitive behaviors. These problems involve mixture-distributed count data that are collected by a quantitative randomized response technique (QRRT) which guarantees respondent anonymity. We propose a Poisson regression method based on maximum likelihood estimation computed via the EM algorithm. This method allows assessment of the importance of potential drivers of different quantities of non-compliant behavior. The method is illustrated with a case study examining potential drivers of non-compliance with hunting regulations in Sierra Leone.
Open Access
The cardiac jelly extracellular matrix contributes to valve development and overall cardiac function
(Colorado State University. Libraries, 2022) Ostwald, Paige, author; Garrity, Deborah, advisor; Bark, David, committee member; Bedinger, Patricia, committee member; Nishimura, Erin, committee member; Peers, Graham, committee member
Nearly 2.6 million infants are born every year with a congenital cardiac anomaly across the entire globe. Congenital heart defects (CHDs) within the valve occur in over 50% of cases. 56% of heart defects have an unknown etiology, illuminating the need for continuous research on heart development and potential causes. Before the valve is a mature structure with established leaflets, the heart forms two endocardial cushions that press together to occlude blood flow between chambers. The cushions are composed of an extracellular matrix called the cardiac jelly (CJ). Previous studies have found evidence of the vital role the cardiac jelly plays within the developing valve for structure, genetic signaling and cell organization. Here, we present a specific role the cardiac jelly plays in valve function and overall cardiac output. To alter the cardiac jelly, we used a morpholino approach in a zebrafish model to increase, decrease and structurally compromise the cardiac jelly. By doing so, we found decreased valve cell differentiation with decreased CJ and increased valve cell differentiation with increased CJ. Using high-speed video technology, we also found decreased valve opening regardless of cardiac jelly alteration, resulting in reduced overall cardiac function. Our results suggest that the function of the endocardial cushions relies on an appropriate presence of CJ. We next investigated just how the cardiac jelly may be altered during development. To do so, we exposed zebrafish embryos to hyperglycemic conditions during the initial and critical heart development period. We found that when embryos absorb over 1.5-fold more D-glucose due to high-glucose conditions, they exhibit significant alterations to CJ width. Altered CJ due to hyperglycemic conditions affected valve differentiation, valve opening, and cardiac function, particularly when embryos have absorbed over a 2-fold increase of glucose. Together, these results show the structural role of the cardiac jelly to support endocardial cushion opening which will supply enough oxygenated blood to the embryo.
Open Access
The role of Ferredoxin 3 in hydrogen metabolism in the hyperthermophilic archaeon Thermococcus kodakarensis
(Colorado State University. Libraries, 2022) Stettler, Meghan, author; Santangelo, Thomas, advisor; Hansen, Jeffrey, committee member; Peers, Graham, committee member
Life faces innumerable challenges to cellular maintenance and reproduction, including access to sufficient energy. As such, all domains of life ubiquitously utilize energetically conservative mechanisms to maximize energy gains from the environment. Use of proteinaceous electron carriers, like ferredoxins, allows cells to harness energy from catabolic reactions that would otherwise be lost to the system as entropy or enthalpy. The hyperthermophilic, anaerobic archaeon Thermococcus kodakarensis is of particular interest as a target for bioengineering to maximize total energy gains, as it natively produces hydrogen gas resulting from terminal electron transport through a Membrane Bound Hydrogenase. T. kodakarensis encodes for three physiologically distinct ferredoxins. Prior to this thesis, only the sequence and molecular weight of the T. kodakarensis ferredoxins were known. Efforts in this thesis laid the groundwork for the biophysical characterization of each ferredoxin isoform via protein-film voltammetry and x-ray crystallography by the development of a reliable recombinant expression and purification scheme. Preliminary biophysical assay trials resulted in a Ferredoxin 1 crystal capable of diffracting to 1.1 Ångstroms, and midpoint reduction potentials for Ferredoxin 1 and Ferredoxin 3 confirming the predicted redox center geometry, demonstrating the efficacy of the developed protein expression and purification scheme for producing high-quality samples. Further investigation into the activity of the ferredoxins resulted in the generation of T. kodakarensis strains encoding for a tether protein between Ferredoxin 3 and its presumed sole electron acceptor Membrane Bound Hydrogenase at two respective locations. The parent strain includes a deletion of Ferredoxin 3, resulting in a deficient phenotype during sulfur-independent growth. The tethered strains of T. kodakarensis demonstrates a full recovery of sulfur-independent growth. Additionally, western-blotting revealed retention of the tethered protein in-vivo, and headspace measurements demonstrated restoration of hydrogen gas production compared to the parent deletion strain, and a reduction in total hydrogen gas output per cell compared to the lab parent strain. These findings implicate the importance of Ferredoxin 3 in hydrogen metabolism in T. kodakarensis and indicate Ferredoxin 3 as a potential target for bioengineering. Furthermore, this thesis is the foundation for further characterization of the T. kodakarensis ferredoxins as proteinaceous electron carriers with potential applications outside of this model organism.
Open Access
Using far-red light to promote leaf expansion for young plant production
(Colorado State University. Libraries, 2022) Percival, Anthony Christophe, author; Craver, Joshua, advisor; Newman, Steven, committee member; Peers, Graham, committee member
At northern latitudes, a reduction in the natural light quantity during the winter production of young annual bedding plants (plugs) often necessitates the use of supplemental lighting to reach a target daily light integral (DLI) to ensure high plug quality. However, the low leaf area index (LAI) of plugs during the initial stages of production suggests that a portion of applied light is not intercepted by leaves. Because electric lighting represents a significant percentage of total production costs for greenhouses utilizing supplemental lighting, minimizing wasted light (photons not absorbed by the plant) is critical. Some species have shown an increase in leaf area in response to growth under light with a low ratio of red to far-red light (R:FR); this is generally considered as a shade avoidance response to improve light capture, but there is considerable variation across species. An early increase in leaf area would allow for more effective light capture by seedlings and a reduction in wasted light, but other shade avoidance responses such as elongation of stems and petioles are undesirable for plug production and could outweigh benefits of leaf expansion. Far-red mediated shade avoidance responses may also depend on background photosynthetic photon flux density, DLI, and temperature. The objective of this research was to investigate the effects of far-red radiation on leaf expansion and other shade avoidance responses for the popular annual bedding plant, Petunia ×hybrida (petunia), and to examine potential influences of other environmental variables. Reducing the R:FR in a greenhouse environment with supplemental lighting is challenging due to the relatively high proportion of natural light, so an end-of-day far-red (EOD-FR) lighting strategy was utilized to investigate the promotion of leaf expansion by far-red light for seedings of petunia 'Wave Purple', and 'Dreams Midnight'. Seedlings were grown in 128-cell trays in a common greenhouse environment under an ambient DLI of 5.26 mols·m-2·d-1 to simulate a winter light environment. Seedlings received no EOD-FR, supplemental lighting for the duration of the experiment, or one of the following EOD-FR treatments that varied in far-red intensity, R:FR ratio, and treatment duration: 10 μmol·m-2·s-1 of far-red light (R:FR ~0.8) for 30 minutes, 10 or 20 μmol·m-2·s-1 of far-red light (R:FR ~0.15) for 30 minutes, or 20 μmol·m-2·s-1 of far-red light (R:FR ~0.15) for 240 minutes. In addition to end-of-day (EOD) treatments, some seedlings under EOD-FR were moved under supplemental lighting after 2 or 3 weeks of EOD lighting. Destructive data was collected 2, 3, and 4 weeks after treatment initiation. Seedlings that received EOD-FR lighting showed stem elongation responses, and seedlings under the lower R:FR or longer EOD duration resulted in greater elongation, but no EOD treatment resulted in an increase in leaf area compared to control (no supplemental lighting or EOD lighting) or supplemental lighting treatments. Results of this study indicate that under low DLIs, EOD-FR light applied in the first three weeks of seedling production does not promote early leaf area expansion and reduces seedling quality under these experimental conditions. To further examine leaf expansion as a response to far-red radiation, seedlings of petunia 'Dreams Midnight' were grown for 28 days under the recommended target DLI of ~10 mols·m-2·d-1 using a 17.25-h photoperiod with either a high (~10.8) or low R:FR (~0.50). The effects of EOD-FR were also examined by subjecting seedlings grown under the high R:FR to a 1-hour low intensity (target total photon flux density of 46 μmol·m2·s-1) EOD lighting application with a very low R:FR (0.15). Lastly, the influence of temperature on the effects of far-red radiation were examined by growing seedlings at either 16 or 21 ℃ for the duration of the experiment, and by moving plants from a high R:FR in the 21 ℃ chamber during the day to the 16 ℃ chamber for the EOD-FR treatment and subsequent dark period. Overall, seedlings grown at a constant air temperature of 16 ℃ displayed stunted growth (lower leaf area, number of leaves, and total biomass) compared to those grown at 21 ℃ regardless of lighting treatment. At 21 ℃, the use of EOD-FR did not promote an increase in leaf area. Seedlings grown under a constant low R:FR (~0.50) at 21 ℃ did display increased leaf area, but lower stem dry mass per unit length (mg·mm-1), leaf mass per unit area (g·m2), and root dry mass indicated poor seedling quality. These results further show that morphological responses to far-red light are species-specific, and that plant responses to far-red light may differ based on a variety of environmental factors. Future research regarding leaf expansion in response to far-red light that incorporates other environmental factors (e.g., temperature, TPFD, photoperiod length) may lead to a more complete understanding of species-specific shade avoidance responses, and further work in this area may assist growers with the development of far-red lighting strategies to improve light capture and seedling quality for young plant production.