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Description
Human activity has increased loading of reactive nitrogen (N) in the environment, with important and often deleterious impacts on biodiversity, climate, and human health. Since the fate of N in the ecosystem is mainly controlled by microorganisms, understanding the factors that shape microbial communities becomes relevant and urgent. In arid land soils, these microbial communities and factors are not well understood. I aimed to study the role of N cycling microbes, such as the ammonia-oxidizing bacteria (AOB), the recently discovered ammonia-oxidizing archaea (AOA), and various fungal groups, in soils of arid lands. I also tested if niche differentiation among microbial populations is a driver of differential biogeochemical outcomes. I found that N cycling microbial communities in arid lands are structured by environmental factors to a stronger degree than what is generally observed in mesic systems. For example, in biological soil crusts, temperature selected for AOA in warmer deserts and for AOB in colder deserts. Land-use change also affects niche differentiation, with fungi being the major agents of N2O production in natural arid lands, whereas emissions could be attributed to bacteria in mesic urban lawns. By contrast, NO3- production in the native desert and managed soils was mainly controlled by autotrophic microbes (i.e., AOB and AOA) rather than by heterotrophic fungi. I could also determine that AOA surprisingly responded positively to inorganic N availability in both short (one month) and long-term (seven years) experimental manipulations in an arid land soil, while environmental N enrichment in other ecosystem types is known to favor AOB over AOA. This work improves our predictions of ecosystem response to anthropogenic N increase and shows that paradigms derived from mesic systems are not always applicable to arid lands. My dissertation also highlights the unique ecology of ammonia oxidizers and draws attention to the importance of N cycling in desert soils.
ContributorsMarusenko, Yevgeniy (Author) / Hall, Sharon J (Thesis advisor) / Garcia-Pichel, Ferran (Thesis advisor) / Mclain, Jean E (Committee member) / Schwartz, Egbert (Committee member) / Arizona State University (Publisher)
Created2013
Description
Microbial electrochemical cells (MXCs) are promising platforms for bioenergy production from renewable resources. In these systems, specialized anode-respiring bacteria (ARB) deliver electrons from oxidation of organic substrates to the anode of an MXC. While much progress has been made in understanding the microbiology, physiology, and electrochemistry of well-studied model ARB such as Geobacter and Shewanella, tremendous potential exists for MXCs as microbiological platforms for exploring novel ARB. This dissertation introduces approaches for selective enrichment and characterization of phototrophic, halophilic, and alkaliphilic ARB. An enrichment scheme based on manipulation of poised anode potential, light, and nutrient availability led to current generation that responded negatively to light. Analysis of phototrophically enriched communities suggested essential roles for green sulfur bacteria and halophilic ARB in electricity generation. Reconstruction of light-responsive current generation could be successfully achieved using cocultures of anode-respiring Geobacter and phototrophic Chlorobium isolated from the MXC enrichments. Experiments lacking exogenously supplied organic electron donors indicated that Geobacter could produce a measurable current from stored photosynthate in the dark. Community analysis of phototrophic enrichments also identified members of the novel genus Geoalkalibacter as potential ARB. Electrochemical characterization of two haloalkaliphilic, non-phototrophic Geoalkalibacter spp. showed that these bacteria were in fact capable of producing high current densities (4-8 A/m2) and using higher organic substrates under saline or alkaline conditions. The success of these selective enrichment approaches and community analyses in identifying and understanding novel ARB capabilities invites further use of MXCs as robust platforms for fundamental microbiological investigations.
ContributorsBadalamenti, Jonathan P (Author) / Krajmalnik-Brown, Rosa (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Rittmann, Bruce E. (Committee member) / Torres, César I (Committee member) / Vermaas, Willem (Committee member) / Arizona State University (Publisher)
Created2013
Description
[FeFe]-hydrogenases are enzymes for the reduction of protons to hydrogen. They rely on only the earth abundant first-row transition metal iron at their active site (H cluster). In recent years, a multitude of diiron mimics of hydrogenases have been synthesized, but none of them catalyzes hydrogen production with the same exquisite combination of high turnover frequency and low activation energy as the enzymes. Generally, model complexes fail to include one or both of two features essential to the natural enzyme: an intricate array of outer coordination sphere contacts that constrain the coordination geometry to attain a catalytically optimal conformation, and the redox non-innocence of accessory [FeS] clusters found at or near the hydrogen-activating site. The work presented herein describes the synthesis and electrocatalytic characterization of iron-dithiolate models designed to incorporate these features. First, synthetic strategies are developed for constructing peptides with artificial metal-binding motifs, such as 1,3-dithiolate and phosphines, which are utilized to append diiron-polycarbonyl clusters onto a peptide. The phosphine-functionalized peptides are shown to be better electrocatalysts for proton reduction in water/acetonitrile mixtures than in neat acetonitrile. Second, we report the impact of redox non-innocent ligands on the electrocatalytic properties of two types of [FeFe]-hydrogenase models: dinuclear and mononuclear iron complexes. The bidentate, redox non-innocent α-diimine ligands (N-N), 2,2'-bipyridine and 2,2' bipyrimidine, are used to create complexes with the general formula (μ-SRS)Fe2(CO)4(N-N), new members of the well known family of asymmetric diiron carbonyls. While the 2,2'-bipyridine derivatives can act as electrocatalysts for proton reduction, surprisingly, the 2,2'-bipyrimidine analogues are found to be inactive towards catalysis. Electrochemical investigation of two related Fe(II) complexes, (bdt)Fe(CO)P2 for bdt = benzene-1,2-dithiolate and P2 = 1,1'-diphenylphosphinoferrocene or methyl-2-{bis(diphenylphosphinomethylamino}acetate, related to the distal iron in [FeFe]-hydrogenase show that these complexes catalyze the reduction of protons under mild conditions. However, their reactivities toward the external ligand CO are distinguished by gross geometrical differences.
ContributorsRoy, Souvik (Author) / Jones, Anne K (Thesis advisor) / Moore, Thomas (Committee member) / Trovitch, Ryan (Committee member) / Arizona State University (Publisher)
Created2013
Description
Thiol functionalization is one potentially useful way to tailor physical and chemical properties of graphene oxides (GOs) and reduced graphene oxides (RGOs). Despite the ubiquitous presence of thiol functional groups in diverse chemical systems, efficient thiol functionalization has been challenging for GOs and RGOs, or for carbonaceous materials in general. In this work, thionation of GOs has been achieved in high yield through two new methods that also allow concomitant chemical reduction/thermal reduction of GOs; a solid-gas metathetical reaction method with boron sulfides (BxSy) gases and a solvothermal reaction method employing phosphorus decasulfide (P4S10). The thionation products, called "mercapto reduced graphene oxides (m-RGOs)", were characterized by employing X-ray photoelectron spectroscopy, powder X-ray diffraction, UV-Vis spectroscopy, FT-IR spectroscopy, Raman spectroscopy, electron probe analysis, scanning electron microscopy, (scanning) transmission electron microscopy, nano secondary ion mass spectrometry, Ellman assay and atomic force microscopy. The excellent dispersibility of m-RGOs in various solvents including alcohols has allowed fabrication of thin films of m-RGOs. Deposition of m-RGOs on gold substrates was achieved through solution deposition and the m-RGOs were homogeneously distributed on gold surface shown by atomic force microscopy. Langmuir-Blodgett (LB) films of m-RGOs were obtained by transferring their Langmuir films, formed by simple drop casting of m-RGOs dispersion on water surface, onto various substrates including gold, glass and indium tin oxide. The m-RGO LB films showed low sheet resistances down to about 500 kΩ/sq at 92% optical transparency. The successful results make m-RGOs promising for applications in transparent conductive coatings, biosensing, etc.
ContributorsJeon, Kiwan (Author) / Seo, Dong-Kyun (Thesis advisor) / Jones, Anne K (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2013
Description
Redox reactions are crucial to energy transduction in biology. Protein film electrochemistry (PFE) is a technique for studying redox proteins in which the protein is immobilized at an electrode surface so as to allow direct exchange of electrons. Establishing a direct electronic connection eliminates the need for redoxactive mediators, thus allowing for interrogation of the redox protein of interest. PFE has proven a versatile tool that has been used to elucidate the properties of many technologically relevant redox proteins including hydrogenases, laccases, and glucose oxidase.
This dissertation is comprised of two parts: extension of PFE to a novel electrode material and application of PFE to the investigation of a new type of hydrogenase. In the first part, mesoporous antimony-doped tin oxide (ATO) is employed for the first time as an electrode material for protein film electrochemistry. Taking advantage of the excellent optical transparency of ATO, spectroelectrochemistry of cytochrome c is demonstrated. The electrochemical and spectroscopic properties of the protein are analogous to those measured for the native protein in solution, and the immobilized protein is stable for weeks at high loadings. In the second part, PFE is used to characterize the catalytic properties of the soluble hydrogenase I from Pyrococcus furiosus (PfSHI). Since this protein is highly thermostable, the temperature dependence of catalytic properties was investigated. I show that the preference of the enzyme for reduction of protons (as opposed to oxidation of hydrogen) and the reactions with oxygen are highly dependent on temperature, and the enzyme is tolerant to oxygen during both oxidative and reductive catalysis.
This dissertation is comprised of two parts: extension of PFE to a novel electrode material and application of PFE to the investigation of a new type of hydrogenase. In the first part, mesoporous antimony-doped tin oxide (ATO) is employed for the first time as an electrode material for protein film electrochemistry. Taking advantage of the excellent optical transparency of ATO, spectroelectrochemistry of cytochrome c is demonstrated. The electrochemical and spectroscopic properties of the protein are analogous to those measured for the native protein in solution, and the immobilized protein is stable for weeks at high loadings. In the second part, PFE is used to characterize the catalytic properties of the soluble hydrogenase I from Pyrococcus furiosus (PfSHI). Since this protein is highly thermostable, the temperature dependence of catalytic properties was investigated. I show that the preference of the enzyme for reduction of protons (as opposed to oxidation of hydrogen) and the reactions with oxygen are highly dependent on temperature, and the enzyme is tolerant to oxygen during both oxidative and reductive catalysis.
ContributorsKwan, Patrick Karchung (Author) / Jones, Anne K (Thesis advisor) / Francisco, Wilson (Committee member) / Moore, Thomas (Committee member) / Arizona State University (Publisher)
Created2014
Description
Increased global demand for energy has led to prolific use of fossil fuels, which produce and release greenhouse gases, such as carbon dioxide. This increase in atmospheric carbon dioxide affects the global weather system and has been cited as a cause for global warming. For humans to continue to meet demands for energy while reducing greenhouse emission, a sustainable, carbon-neutral energy source must be developed. The sun provides energy for the majority of life on earth, as well as the energy stored in the chemical bonds of fossil fuels. This dissertation investigates systems inspired by the biological mechanism of solar energy capture and storage. In natural photosynthesis, organisms use chlorophyll as a chromophore to absorb the sun's energy. Bio-inspired systems use close analogues like porphyrins and phthalocyanines. In this dissertation, a soluble, semiconducting porphyrin is reported. The polymer was synthesized via a Buchwald-Hartwig style coupling of porphyrin monomers which produced a polyaniline-like chain with porphyrins incorporated into the backbone. Spectroscopic and electrochemical studies were performed, which show evidence of excited state charge transfer and a first oxidation state of 0.58 V (vs SCE). These properties suggest that the polymer could be involved in excited state electron donation to fullerenes and other electron acceptors, which could be beneficial in organic photovoltaics, sensors, and other applications. Molecular dyads and triads capable of charge separation have been studied for decades, and the spectroscopic properties of two novel systems are reported in this dissertation. A peripherally-connected zinc-phthalocyanine-C60 dyad was studied, and showed excited state electron transfer from the phthalocyanine excited state to the C60, with a long-lived charge separated state. An axially-linked carotene-Si-pthalocyanine-C60 triad was studied, showing excited state electron transfer from the phthalocyanine to the C60, but fast recombination before hole transfer can occur to the carotene. Analogues of the electron transport mechanisms used in many biological systems use iron-sulfur clusters to shuttle electrons from donors to acceptors. In this dissertation, the spectroscopic properties of a de novo protein were studied. Nanosecond transient absorption was used to characterize the electron and energy transfer of an excited water-soluble porphyrin to the oxidized [FeS] clusters incorporated in the de novo protein. The triplet state of the porphyrin was strongly quenched with the holo-protein without a rise in porphyrin plus signal, suggesting that only Dexter-type energy transfer occurs between the sensitized porphyrin and the [FeS] clusters.
ContributorsSchmitz, Robert (Author) / Gust, John D (Thesis advisor) / Jones, Anne K (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2014
Description
Molybdenum (Mo) is a key trace nutrient for biological assimilation of nitrogen, either as nitrogen gas (N2) or nitrate (NO3-). Although Mo is the most abundant metal in seawater (105 nM), its concentration is low (<5 nM) in most freshwaters today, and it was scarce in the ocean before 600 million years ago. The use of Mo for nitrogen assimilation can be understood in terms of the changing Mo availability through time; for instance, the higher Mo content of eukaryotic vs. prokaryotic nitrate reductase may have stalled proliferation of eukaryotes in low-Mo Proterozoic oceans. Field and laboratory experiments were performed to study Mo requirements for NO3- assimilation and N2 fixation, respectively. Molybdenum-nitrate addition experiments at Castle Lake, California revealed interannual and depth variability in plankton community response, perhaps resulting from differences in species composition and/or ammonium availability. Furthermore, lake sediments were elevated in Mo compared to soils and bedrock in the watershed. Box modeling suggested that the largest source of Mo to the lake was particulate matter from the watershed. Month-long laboratory experiments with heterocystous cyanobacteria (HC) showed that <1 nM Mo led to low N2 fixation rates, while 10 nM Mo was sufficient for optimal rates. At 1500 nM Mo, freshwater HC hyperaccumulated Mo intercellularly, whereas coastal HC did not. These differences in storage capacity were likely due to the presence in freshwater HC of the small molybdate-binding protein, Mop, and its absence in coastal and marine cyanobacterial species. Expression of the mop gene was regulated by Mo availability in the freshwater HC species Nostoc sp. PCC 7120. Under low Mo (<1 nM) conditions, mop gene expression was up-regulated compared to higher Mo (150 and 3000 nM) treatments, but the subunit composition of the Mop protein changed, suggesting that Mop does not bind Mo in the same manner at <1 nM Mo that it can at higher Mo concentrations. These findings support a role for Mop as a Mo storage protein in HC and suggest that freshwater HC control Mo cellular homeostasis at the post-translational level. Mop's widespread distribution in prokaryotes lends support to the theory that it may be an ancient protein inherited from low-Mo Precambrian oceans.
ContributorsGlass, Jennifer (Author) / Anbar, Ariel D (Thesis advisor) / Shock, Everett L (Committee member) / Jones, Anne K (Committee member) / Hartnett, Hilairy E (Committee member) / Elser, James J (Committee member) / Fromme, Petra (Committee member) / Arizona State University (Publisher)
Created2011
Description
The oceans play an essential role in global biogeochemical cycles and in regulating climate. The biological carbon pump, the photosynthetic fixation of carbon dioxide by phytoplankton and subsequent sequestration of organic carbon into deep water, combined with the physical carbon pump, make the oceans the only long-term net sink for anthropogenic carbon dioxide. A full understanding of the workings of the biological carbon pump requires a knowledge of the role of different taxonomic groups of phytoplankton (protists and cyanobacteria) to organic carbon export. However, this has been difficult due to the degraded nature of particles sinking into particle traps, the main tools employed by oceanographers to collect sinking particulate matter in the ocean. In this study DNA-based molecular methods, including denaturing gradient gel electrophoresis, cloning and sequencing, and taxon-specific quantitative PCR, allowed for the first time for the identification of which protists and cyanobacteria contributed to the material collected by the traps in relation to their presence in the euphotic zone. I conducted this study at two time-series stations in the subtropical North Atlantic Ocean, one north of the Canary Islands, and one located south of Bermuda. The Bermuda study allowed me to investigate seasonal and interannual changes in the contribution of the plankton community to particle flux. I could also show that small unarmored taxa, including representatives of prasinophytes and cyanobacteria, constituted a significant fraction of sequences recovered from sediment trap material. Prasinophyte sequences alone could account for up to 13% of the clone library sequences of trap material during bloom periods. These observations contradict a long-standing paradigm in biological oceanography that only large taxa with mineral shells are capable of sinking while smaller, unarmored cells are recycled in the euphotic zone through the microbial loop. Climate change and a subsequent warming of the surface ocean may lead to a shift in the protist community toward smaller cell size in the future, but in light of these findings these changes may not necessarily lead to a reduction in the strength of the biological carbon pump.
ContributorsAmacher, Jessica (Author) / Neuer, Susanne (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Lomas, Michael (Committee member) / Wojciechowski, Martin (Committee member) / Stout, Valerie (Committee member) / Arizona State University (Publisher)
Created2011
Description
Biological soil crusts (BSCs) are critical components of arid and semiarid environments and provide the primary sources of bioavailable macronutrients and increase micronutrient availability to their surrounding ecosystems. BSCs are composed of a variety of microorganisms that perform a wide range of physiological processes requiring a multitude of bioessential micronutrients, such as iron, copper, and molybdenum. This work investigated the effects of BSC activity on soil solution concentrations of bioessential elements and examined the microbial production of organic chelators, called siderophores. I found that aluminum, vanadium, copper, zinc, and molybdenum were solubilized in the action of crusts, while nickel, zinc, arsenic, and zirconium were immobilized by crust activity. Potassium and manganese displayed behavior consistent with biological removal and mobilization, whereas phosphorus and iron solubility were dominated by abiotic processes. The addition of bioavailable nitrogen altered the effects of BSCs on soil element mobilization. In addition, I found that the biogeochemical activites of BSCs were limited by molybdenum, a fact that likely contributes to co-limitation by nitrogen. I confirmed the presence of siderophore producing microbes in BSCs. Siderophores are low-molecular weight organic compounds that are released by bacteria to increase element solubility and facilitate element uptake; siderophore production is likely the mechanism by which BSCs affect the patterns I observed in soil solution element concentrations. Siderophore producers were distributed across a range of bacterial groups and ecological niches within crusts, suggesting that siderophore production influences the availability of a variety of elements for use in many physiological processes. Four putative siderophore compounds were identified using electrospray ionization mass spectrometry; further attempts to characterize the compounds confirmed two true siderophores. Taken together, the results of my work provide information about micronutrient cycling within crusts that can be applied to BSC conservation and management. Fertilization with certain elements, particularly molybdenum, may prove to be a useful technique to promote BSC growth and development which would help prevent arid land degradation. Furthermore, understanding the effects of BSCs on soil element mobility could be used to develop useful biomarkers for the study of the existence and distribution of crust-like communities on ancient Earth, and perhaps other places, like Mars.
ContributorsNoonan, Kathryn Alexander (Author) / Hartnett, Hilairy (Thesis advisor) / Anbar, Ariel (Committee member) / Garcia-Pichel, Ferran (Committee member) / Shock, Everett (Committee member) / Sharp, Thomas (Committee member) / Arizona State University (Publisher)
Created2012
Description
Hydrogenases, the enzymes that reversibly convert protons and electrons to hydrogen, are used in all three domains of life. [NiFe]-hydrogenases are considered best suited for biotechnological applications because of their reversible inactivation with oxygen. Phylogenetically, there are four groups of [NiFe]-hydrogenases. The best characterized group, "uptake" hydrogenases, are membrane-bound and catalyze hydrogen oxidation in vivo. In contrast, the group 3 [NiFe]-hydrogenases are heteromultimeric, bifunctional enzymes that fulfill various cellular roles. In this dissertation, protein film electrochemistry (PFE) is used to characterize the catalytic properties of two group 3 [NiFe]-hydrogenases: HoxEFUYH from Synechocystsis sp. PCC 6803 and SHI from Pyrococcus furiosus. First, HoxEFUYH is shown to be biased towards hydrogen production. Upon exposure to oxygen, HoxEFUYH inactivates to two states, both of which can be reactivated on the timescale of seconds. Second, we show that PfSHI is the first example of an oxygen tolerant [NiFe]-hydrogenase that produces two inactive states upon exposure to oxygen. Both inactive states are analogous to those characterized for HoxEFUYH, but oxygen exposed PfSHI produces a greater fraction that reactivates at high potentials, enabling hydrogen oxidation in the presence of oxygen. Third, it is shown that removing the NAD(P)-reducing subunits from PfSHI leads to a decrease in bias towards hydrogen oxidation and renders the enzyme oxygen sensitive. Both traits are likely due to impaired intramolecular electron transfer. Mechanistic hypotheseses for these functional differences are considered.
ContributorsMcIntosh, Chelsea Lee (Author) / Jones, Anne K (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2012