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The ocean sequesters more than 25% of the carbon released by anthropogenic action every year, and oligotrophic oceans, such as the Sargasso Sea, are responsible for about 50% of the global carbon export. Pico- and nano-phytoplankton (cells < 5 µm), mostly unicellular eukaryotes (protists) and cyanobacteria, dominate the primary production in the Sargasso Sea; however, little is known about their contribution to the export of carbon into the deep ocean via sinking particles. The overall goal of this study is to examine the link between growth and grazing rates of pico- and nano-phytoplankton and the carbon export in the Sargasso Sea. I investigate three aspects: 1) how microzooplankton grazing and physical forcing affect taxon-specific primary productivity in this region, 2) how these microbial trophic dynamics impact their contribution to the export of particulate matter, and 3) how much pico-phytoplankton, specifically the pico-cyanobacteria Synechococcus and Prochlorococcus, contribute to the carbon export. I collected seawater samples within the sunlit (euphotic) zone, and sinking particles at 150 m depth using particle traps in the Sargasso Sea during the winter and summer seasons of 2011 and 2012. I conducted dilution experiments to determine the growth and grazing rates of the pico- and nano-phytoplankton community, and used 454 pyrosequencing and quantitative Polymerase Chain Reaction to measure the relative and absolute contribution of these primary producers to the plankton community within the euphotic zone and in the sinking particles. I found that micrograzing controls taxon-specific primary production, and that microbial trophic dynamics impact directly the taxonomical composition of the sinking particles. For the first time, I was able to quantify clade-specific carbon export of pico-cyanobacteria and found that, despite their small size, these tiny primary producers are capable of sinking from the surface to the deeper oceans. However, their contribution to the carbon flux is often less than one tenth of their biomass contribution in the euphotic zone. Our study provides a comprehensive approach to better understand the role of pico- and nano-phytoplankton in the carbon cycle of oligotrophic oceans, and a baseline to study changes in the carbon export in future warmer oceans.
ContributorsDe Martini, Francesca (Author) / Neuer, Susanne (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Hartnett, Hilairy (Committee member) / Lomas, Michael (Committee member) / Arizona State University (Publisher)
Created2016
Description
The aboveground surfaces of plants (i.e. the phyllosphere) comprise the largest biological interface on Earth (over 108 km2). The phyllosphere is a diverse microbial environment where bacterial inhabitants have been shown to sequester and degrade airborne pollutants (i.e. phylloremediation). However, phyllosphere dynamics are not well understood in urban environments, and this environment has never been studied in the City of Phoenix, which maintains roughly 92,000 city trees. The phyllosphere will grow if the City of Phoenix is able to achieve its goal of 25% canopy coverage by 2030, but this begs the question: How and where should the urban canopy expand? I addressed this question from a phyllosphere perspective by sampling city trees of two species, Ulmus parvifolia (Chinese Elm) and Dalbergia sissoo (Indian Rosewood) in parks and on roadsides. I identified characteristics of the bacterial community structure and interpreted the ecosystem service potential of trees in these two settings. I used culture-independent methods to compare the abundance of each unique bacterial lineage (i.e. ontological taxonomic units or OTUs) on the leaves of park trees versus on roadside tree leaves. I found numerous bacteria (81 OTUs) that were significantly more abundant on park trees than on roadside trees. Many of these OTUs are ubiquitous to bacterial phyllosphere communities, are known to promote the health of the host tree, or have been shown to degrade airborne pollutants. Roadside trees had fewer bacteria (10 OTUs) that were significantly more abundant when compared to park trees, but several have been linked to the remediation of petroleum combustion by-products. These findings, that were not available prior to this study, may inform the City of Phoenix as it is designing its future urban forests.
ContributorsMacNeille, Benjamin C (Author) / Childers, Daniel L. (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Cease, Arianne J (Committee member) / Arizona State University (Publisher)
Created2016
Description
The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains a NiFe-type bidirectional hydrogenase that is capable of using reducing equivalents to reduce protons and generate H¬2. In order to achieve sustained H2 production using this cyanobacterium many challenges need to be overcome. Reported H2 production from Synechocystis is of low rate and often transient. Results described in this dissertation show that the hydrogenase activity in Synechocystis is quite different during periods of darkness and light. In darkness, the hydrogenase enzyme acts in a truly bidirectional way and a particular H2 concentration is reached that depends upon the amount of biomass involved in H2 production. On the other hand, in the presence of light the enzyme shows only transient H2 production followed by a rapid and constitutive H2 oxidation. H2 oxidation and production were measured from a variety of Synechocystis strains in which components of the photosynthetic or respiratory electron transport chain were either deleted or inhibited. It was shown that the light-induced H2 oxidation is dependent on the activity of cytochrome b6f and photosystem I but not on the activity of photosystem II, indicating a channeling of electrons through cytochrome b6f and photosystem I. Because of the sequence similarities between subunits of NADH dehydrogenase I in E. coli and subunits of hydrogenase in Synechocystis, NADH dehydrogenase I was considered as the most likely candidate to mediate the electron transfer from hydrogenase to the membrane electron carrier plastoquinone, and a three-dimensional homology model with the associated subunits shows that structurally it is possible for the subunits of the two complexes to assemble. Finally, with the aim of improving the rate of H2 production in Synechocystis by using a powerful hydrogenase enzyme, a mutant strain of Synechocystis was created in which the native hydrogenase was replaced with the hydrogenase from Lyngbya aestuarii BL J, a strain with higher capacity for H2 production. H2 production was detected in this Synechocystis mutant strain, but only in the presence of external reductants. Overall, this study emphasizes the importance of redox partners in determining the direction of H2 flux in Synechocystis.
ContributorsDatta, Īpsitā (Author) / Vermaas, Willem Fj (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Rittmann, Bruce (Committee member) / Jones, Anne K (Committee member) / Arizona State University (Publisher)
Created2015
Description
Many acidic hot springs in Yellowstone National Park support microbial iron oxidation, reduction, or microbial iron redox cycling (MIRC), as determined by microcosm rate experiments. Microbial dissimilatory iron reduction (DIR) was detected in numerous systems with a pH < 4. Rates of DIR are influenced by the availability of ferric minerals and organic carbon. Microbial iron oxidation (MIO) was detected from pH 2 – 5.5. In systems with abundant Fe (II), dissolved oxygen controls the presence of MIO. Rates generally increase with increased Fe(II) concentrations, but rate constants are not significantly altered by additions of Fe(II). MIRC was detected in systems with abundant ferric mineral deposition.
The rates of microbial and abiological iron oxidation were determined in a variety of cold (T= 9-12°C), circumneutral (pH = 5.5-9) environments in the Swiss Alps. Rates of MIO were measured in systems up to a pH of 7.4; only abiotic processes were detected at higher pH values. Iron oxidizing bacteria (FeOB) were responsible for 39-89% of the net oxidation rate at locations where biological iron oxidation was detected. Members of putative iron oxidizing genera, especially Gallionella, are abundant in systems where MIO was measured. Speciation calculations reveal that ferrous iron typically exists as FeCO30, FeHCO3+, FeSO40 or Fe2+ in these systems. The presence of ferrous (bi)carbonate species appear to increase abiotic iron oxidation rates relative to locations without significant concentrations. This approach, integrating geochemistry, rates, and community composition, reveals biogeochemical conditions that permit MIO, and locations where the abiotic rate is too fast for the biotic process to compete.
For a reaction to provide habitability for microbes in a given environment, it must energy yield and this energy must dissipate slowly enough to remain bioavailable. Thermodynamic boundaries exist at conditions where reactions do not yield energy, and can be quantified by calculations of chemical energy. Likewise, kinetic boundaries exist at conditions where the abiotic reaction rate is so fast that reactants are not bioavailable; this boundary can be quantified by measurements biological and abiological rates. The first habitability maps were drawn, using iron oxidation as an example, by quantifying these boundaries in geochemical space.
The rates of microbial and abiological iron oxidation were determined in a variety of cold (T= 9-12°C), circumneutral (pH = 5.5-9) environments in the Swiss Alps. Rates of MIO were measured in systems up to a pH of 7.4; only abiotic processes were detected at higher pH values. Iron oxidizing bacteria (FeOB) were responsible for 39-89% of the net oxidation rate at locations where biological iron oxidation was detected. Members of putative iron oxidizing genera, especially Gallionella, are abundant in systems where MIO was measured. Speciation calculations reveal that ferrous iron typically exists as FeCO30, FeHCO3+, FeSO40 or Fe2+ in these systems. The presence of ferrous (bi)carbonate species appear to increase abiotic iron oxidation rates relative to locations without significant concentrations. This approach, integrating geochemistry, rates, and community composition, reveals biogeochemical conditions that permit MIO, and locations where the abiotic rate is too fast for the biotic process to compete.
For a reaction to provide habitability for microbes in a given environment, it must energy yield and this energy must dissipate slowly enough to remain bioavailable. Thermodynamic boundaries exist at conditions where reactions do not yield energy, and can be quantified by calculations of chemical energy. Likewise, kinetic boundaries exist at conditions where the abiotic reaction rate is so fast that reactants are not bioavailable; this boundary can be quantified by measurements biological and abiological rates. The first habitability maps were drawn, using iron oxidation as an example, by quantifying these boundaries in geochemical space.
ContributorsSt Clair, Brian (Author) / Shock, Everett L (Thesis advisor) / Anbar, Ariel (Committee member) / Garcia-Pichel, Ferran (Committee member) / Hartnett, Hilairy (Committee member) / Arizona State University (Publisher)
Created2017
Description
Immunosignature is a technology that retrieves information from the immune system. The technology is based on microarrays with peptides chosen from random sequence space. My thesis focuses on improving the Immunosignature platform and using Immunosignatures to improve diagnosis for diseases. I first contributed to the optimization of the immunosignature platform by introducing scoring metrics to select optimal parameters, considering performance as well as practicality. Next, I primarily worked on identifying a signature shared across various pathogens that can distinguish them from the healthy population. I further retrieved consensus epitopes from the disease common signature and proposed that most pathogens could share the signature by studying the enrichment of the common signature in the pathogen proteomes. Following this, I worked on studying cancer samples from different stages and correlated the immune response with whether the epitope presented by tumor is similar to the pathogen proteome. An effective immune response is defined as an antibody titer increasing followed by decrease, suggesting elimination of the epitope. I found that an effective immune response usually correlates with epitopes that are more similar to pathogens. This suggests that the immune system might occupy a limited space and can be effective against only certain epitopes that have similarity with pathogens. I then participated in the attempt to solve the antibiotic resistance problem by developing a classification algorithm that can distinguish bacterial versus viral infection. This algorithm outperforms other currently available classification methods. Finally, I worked on the concept of deriving a single number to represent all the data on the immunosignature platform. This is in resemblance to the concept of temperature, which is an approximate measurement of whether an individual is healthy. The measure of Immune Entropy was found to work best as a single measurement to describe the immune system information derived from the immunosignature. Entropy is relatively invariant in healthy population, but shows significant differences when comparing healthy donors with patients either infected with a pathogen or have cancer.
ContributorsWang, Lu (Author) / Johnston, Stephen (Thesis advisor) / Stafford, Phillip (Committee member) / Buetow, Kenneth (Committee member) / McFadden, Grant (Committee member) / Arizona State University (Publisher)
Created2018
Description
Biological soil crusts (biocrust) are photosynthetic communities of organisms forming in the top millimeters of unvegetated soil. Because soil crusts contribute several ecosystem services to the areas they inhabit, their loss under anthropogenic pressure has negative ecological consequences. There is a considerable interest in developing technologies for biocrust restoration such as biocrust nurseries to grow viable inoculum and the optimization of techniques for field deployment of this inoculum. For the latter, knowledge of the natural rates of biocrust dispersal is needed. Lateral dispersal can be based on self-propelled motility by component microbes, or on passive transport through propagule entrainment in runoff water or wind currents, all of which remain to be assessed. I focused my research on determining the capacity of biocrust for lateral self-propelled dispersal. Over the course of one year, I set up two greenhouse experiments where sterile soil substrates were inoculated with biocrusts and where the lateral advancement of biocrust and their cyanobacteria was monitored using time-course photography, discrete determination of soil chlorophyll a concentration, and microscopic observations. Appropriate uninoculated controls were also set up and monitored. These experiments confirm that cyanobacterial biological soil crusts are capable of laterally expanding when provided with presumably optimal watering regime similar to field conditions and moderate temperatures. The maximum temperatures of Sonoran Desert summer (up to 42 °C), exacerbated in the greenhouse setting (48 °C), caused a loss of biomass and the cessation of lateral dispersal, which resumed as temperature decreased. In 8 independent experiments, biocrust communities advanced laterally at an average rate of 2 cm per month, which is half the maximal rate possible based on the instantaneous speed of gliding motility of the cyanobacterium Microcoleus vaginatus. In a span of three months, populations of M. vaginatus, M. steenstrupii, and Scytonema spp. advanced 1 cm/month on average. The advancing crust front was found to be preferentially composed of hormogonia (differentiated, fast-gliding propagules of cyanobacteria). Having established the potential for laterally self-propelled community dispersal (without wind or runoff contributions) will help inform restoration efforts by proposing minimal inoculum size and optimal distance between inoculum patches.
ContributorsSorochkina, Kira (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Rowe, Helen (Committee member) / Wu, Jianguo (Committee member) / Arizona State University (Publisher)
Created2017
Description
Decay of plant litter represents an enormous pathway for carbon (C) into the atmosphere but our understanding of the mechanisms driving this process is particularly limited in drylands. While microbes are a dominant driver of litter decay in most ecosystems, their significance in drylands is not well understood and abiotic drivers such as photodegradation are commonly perceived to be more important. I assessed the significance of microbes to the decay of plant litter in the Sonoran Desert. I found that the variation in decay among 16 leaf litter types was correlated with microbial respiration rates (i.e. CO2 emission) from litter, and rates were strongly correlated with water-vapor sorption rates of litter. Water-vapor sorption during high-humidity periods activates microbes and subsequent respiration appears to be a significant decay mechanism. I also found that exposure to sunlight accelerated litter decay (i.e. photodegradation) and enhanced subsequent respiration rates of litter. The abundance of bacteria (but not fungi) on the surface of litter exposed to sunlight was strongly correlated with respiration rates, as well as litter decay, implying that exposure to sunlight facilitated activity of surface bacteria which were responsible for faster decay. I also assessed the response of respiration to temperature and moisture content (MC) of litter, as well as the relationship between relative humidity and MC. There was a peak in respiration rates between 35-40oC, and, unexpectedly, rates increased from 55 to 70oC with the highest peak at 70oC, suggesting the presence of thermophilic microbes or heat-tolerant enzymes. Respiration rates increased exponentially with MC, and MC was strongly correlated with relative humidity. I used these relationships, along with litter microclimate and C loss data to estimate the contribution of this pathway to litter C loss over 34 months. Respiration was responsible for 24% of the total C lost from litter – this represents a substantial pathway for C loss, over twice as large as the combination of thermal and photochemical abiotic emission. My findings elucidate two mechanisms that explain why microbial drivers were more significant than commonly assumed: activation of microbes via water-vapor sorption and high respiration rates at high temperatures.
ContributorsTomes, Alexander (Author) / Day, Thomas (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Ball, Becky (Committee member) / Hall, Sharon (Committee member) / Roberson, Robert (Committee member) / Arizona State University (Publisher)
Created2020
Description
Desert organisms lead harsh lives owing to the extreme, often unpredictable environmental conditions they endure. Climate change will likely make their existence even harsher. Predicting the ecological consequences of future climate scenarios thus requires understanding how the biota will be affected by climatic shifts. Biological soil crusts (biocrusts) are an important ecosystem component in arid lands, one that covers large portions of the landscape, improving soil stability and fertility. Because cyanobacteria are biocrust’s preeminent primary producers, eking out an existence during short pulses of precipitation, they represent a relevant global change object of study. I assessed how climate scenarios predicted for the Southwestern United States (US) will affect biocrusts using long-term, rainfall-modifying experimental set-ups that imposed either more intense drought, a seasonally delayed monsoon season, or a shift to smaller but more frequent precipitation events. I expected drought to be detrimental, but not a delay in the monsoon season. Surprisingly, both treatments showed similar effects on cyanobacterial community composition and population size after four years. While successionally incipient biocrusts were unaffected, mature biocrusts lost biomass and diversity with treatment, especially among nitrogen-fixing cyanobacteria. In separate experiments, I assessed the effect of rainfall with modified event size and frequency after a decade of treatment. Small, frequent rainfall events surprisingly enhanced the diversity and biomass of bacteria and cyanobacteria, with clear winners and losers: nitrogen-fixing Scytonema sp. benefited, while Microcoleus vaginatus lost its dominance. As an additional finding, I could also show that water addition is not always beneficial to biocrusts, calling into question the notion that these are strictly water-limited systems.
Finally, results interpretation was severely hampered by a lack of appropriate systematic treatment for an important group of biocrust cyanobacteria, the “Microcoleus steenstrupii complex”. I characterized the complex using a polyphasic approach, leading to the formal description of a new family (Porphyrosiphonaceae) of desiccation resistant cyanobacteria that includes 11 genera, of which 5 had to be newly described. Under the new framework, the distribution and abundance of biocrust cyanobacteria with respect to environmental conditions can now be understood. This body of work contributes significantly to explain current distributional patterns of biocrust cyanobacteria and to predict their fate in the face of climate change.
Finally, results interpretation was severely hampered by a lack of appropriate systematic treatment for an important group of biocrust cyanobacteria, the “Microcoleus steenstrupii complex”. I characterized the complex using a polyphasic approach, leading to the formal description of a new family (Porphyrosiphonaceae) of desiccation resistant cyanobacteria that includes 11 genera, of which 5 had to be newly described. Under the new framework, the distribution and abundance of biocrust cyanobacteria with respect to environmental conditions can now be understood. This body of work contributes significantly to explain current distributional patterns of biocrust cyanobacteria and to predict their fate in the face of climate change.
ContributorsMoreira Camara Fernandes, Vanessa (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Rudgers, Jennifer (Committee member) / Sala, Osvaldo (Committee member) / Penton, Christopher (Committee member) / Arizona State University (Publisher)
Created2020
Description
This work advances structural and biophysical studies of three proteins important in disease. First protein of interest is the Francisella tularensis outer membrane protein A (FopA), which is a virulence determinant of tularemia. This work describes recombinant expression in Escherichia coli and successful purification of membrane translocated FopA. The purified protein was dimeric as shown by native polyacrylamide gel electrophoresis and small angle X-ray scattering (SAXS) analysis, with an abundance of β-strands based on circular dichroism spectroscopy. SAXS data supports the presence of a pore. Furthermore, protein crystals of membrane translocated FopA were obtained with preliminary X-ray diffraction data. The identified crystallization condition provides the means towards FopA structure determination; a valuable tool for structure-based design of anti-tularemia therapeutics.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
Next, the nonstructural protein μNS of avian reoviruses was investigated using in vivo crystallization and serial femtosecond X-ray crystallography. Avian reoviruses infect poultry flocks causing significant economic losses. μNS is crucial in viral factory formation facilitating viral replication within host cells. Thus, structure-based targeting of μNS has the potential to disrupt intracellular viral propagation. Towards this goal, crystals of EGFP-tagged μNS (EGFP-μNS (448-605)) were produced in insect cells. The crystals diffracted to 4.5 Å at X-ray free electron lasers using viscous jets as crystal delivery methods and initial electron density maps were obtained. The resolution reported here is the highest described to date for μNS, which lays the foundation towards its structure determination.
Finally, structural, and functional studies of human Threonine aspartase 1 (Taspase1) were performed. Taspase1 is overexpressed in many liquid and solid malignancies. In the present study, using strategic circular permutations and X-ray crystallography, structure of catalytically active Taspase1 was resolved. The structure reveals the conformation of a 50 residues long fragment preceding the active side residue (Thr234), which has not been structurally characterized previously. This fragment adopted a straight helical conformation in contrast to previous predictions. Functional studies revealed that the long helix is essential for proteolytic activity in addition to the active site nucleophilic residue (Thr234) mediated proteolysis. Together, these findings enable a new approach for designing anti-cancer drugs by targeting the long helical fragment.
ContributorsNagaratnam, Nirupa (Author) / Fromme, Petra (Thesis advisor) / Johnston, Stephen (Thesis advisor) / Van Horn, Wade (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
Modern agriculture faces multiple challenges: it must produce more food for a growing global population, adopt more efficient and sustainable management strategies, and adapt to climate change. One potential component of a sustainable management strategy is the application of biochar to agricultural soils. Biochar is the carbon-rich product of biomass pyrolysis, which contains large proportions of aromatic compounds that influence its stability in soil. Concomitant with carbon sequestration, biochar has the potential to increase soil fertility through increasing soil pH, moisture and nutrient retention. Changes in the soil physical and chemical properties can result in shifts in the soil microbiome, which are the proximate drivers of soil processes. This dissertation aims to determine the compositional and functional changes in the soil microbial community in response to the addition of a low-volatile matter biochar. First, the impact of biochar on the bacterial community was investigated in two important agricultural soils (Oxisol and Mollisol) with contrasting fertility under two different cropping systems (conventional sweet corn and zero-tillage napiergrass) one month and one year after the initial addition. This study revealed that the effects of biochar on the bacterial community were most pronounced in the Oxisol under napiergrass cultivation, however soil type was the strongest determinant of the bacterial community. A follow-up study was conducted using shotgun metagenomics to probe the functional community of soil microcosms, which contained Oxisol soil under napiergrass two years after the initial addition of biochar. Biochar significantly increased total carbon in the soils but had little impact on other soil properties. Theses analyses showed that biochar-amended soil microcosms exhibited significant shifts in the functional community and key metabolic pathways related to carbon turnover and denitrification. Given the distinct alterations to the biochar-amended community, deoxyribose nucleic acid (DNA) stable isotope probing was used to target the active populations. These analyses revealed that biochar did not significantly shift the active community in soil microcosms. Overall, these results indicate that the impact of biochar on the active soil community is transient in nature. Yet, biochar may still be a promising strategy for long-term carbon sequestration in agricultural soils.
ContributorsYu, Julian (Author) / Penton, C. Ryan (Thesis advisor) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Hall, Sharon (Committee member) / Arizona State University (Publisher)
Created2020