Interactions between reactive nitrogen and manganese: Implications for marine nitrous oxide cycling

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Cavazos, Amanda Rae
Glass, Jennifer B.
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Nitrous oxide (N2O) is a potent greenhouse gas that can destroy stratospheric ozone. Production and consumption of N2O has long been assumed to be controlled solely by nitrogen-metabolizing microbes. It has recently been shown, however, that intermediate metabolites from these microbes can potentially leak out of cells and react with metal oxides to produce N2O. Typically, these interactions are assumed to occur with iron (Fe) oxides. Recent studies have shown that biotic-abiotic coupling can also occur with manganese (Mn) oxides, especially when reduced by the nitrification intermediate hydroxylamine (NH2OH). Like Fe oxides, Mn oxides are ubiquitous in sediments and at redox interfaces. Nitrous oxide production from NH2OH oxidation occurs more rapidly with Mn oxides than with Fe oxides, yet little is known about the mechanisms or relevance of these interactions in marine systems. To better constrain the importance of nitrogenous intermediates-Mn interactions in the oceans, it is essential to characterize the spatial and temporal nature of N-Mn oxide interactions. This dissertation aims to constrain the importance of coupled biotic/abiotic interactions between reactive nitrogenous intermediates and manganese oxides in marine systems by (1) characterizing the kinetics of NH2OH oxidation by an environmentally-relevant Mn oxide (Ch. 2), (2) developing a rapid, readily-available, and cost-effective method to visualize associations of microbes and manganese oxide particles (Ch. 3), and (3) developing a method to more rapidly and accurately measure NH2OH in water samples (Ch. 4). Previous studies characterized NH2OH oxidation by high oxidation state Mn oxides in conditions that could not be considered relevant to natural environments. My research has focused on developing rate laws and constants relevant for N2O emission models. First, I characterized the kinetics of NH2OH chemo-oxidation, or the abiotic oxidation of NH2OH, by birnessite, a Mn oxide commonly found in the environment. Hydroxylamine was found to rapidly and completely chemo-oxidize to N2O in synthetic ocean water at circumneutral pH (6.2 < pH < 8.3) (Ch. 2). Complete conversion of NH2OH to N2O occurred within 3 min in all experimental runs. The reaction was overall first order with a rate constant of 0.01 s-1. I propose that N2O is produced via a two electron transfer from NH2OH to a Mn(IV) center, forming aminoxyl radical during the first electron transfer and nitroxyl (HNO) during the second transfer. The adsorption of HNO on the excess birnessite surface is predicted to slow the rate of HNO dimerization to N2O, making it the rate-limiting step. Thus, the experimentally derived rate law suggests that NH2OH chemo-oxidation could be a relevant source of marine N2O emissions and should be included in future studies. Building on my finding that NH2OH chemo-oxidation completely and rapidly produces N2O, I then developed a rapid, cost-effective, and readily available method that determines the possibility of NH2OH chemo-oxidation occurring in natural environments. There remains uncertainty as to whether ammonia-oxidizing microbes, which produce NH2OH, associate with Mn oxides in marine or any environments. Current methods that determine the co-localization of microbes with minerals are either not readily available, are expensive, require extensive sample preparation, or require long wait times for equipment use. In Chapter 3, I present a novel method that uses differential interference contrast (DIC) and epifluorescent microscopy in tandem to determine the co-localization of microbes with Mn(III/IV) oxide particles on filtered environmental samples on white filters. Filters are stained with the Mn-specific stain leucoberbelin blue (LBB) to create characteristic blue haloes or imprints around Mn oxide particles followed by staining with the fluorescent nucleic acid stain SYBR Green. Mn oxide particles are imaged using DIC microscopy and cells are then imaged using a fluorescent SYBR Green (excitation: 395 nm/emission: 509 nm) light set. Manganese oxide identification and overlay of SYBR Green image is done using the image software ImageJ using color threshold and overlay functions. This method was successfully applied to laboratory and environmental samples and has the potential to be used as a rapid, cost-effective “pre-screen” to determine which samples are worth the time and money for higher resolution imaging. Thus, the significance of Mn oxides in marine biogeochemical cycles can be quickly and effectively studied. While the role of NH2OH in terrestrial N2O emissions has been studied in recent years, measurements in marine systems are lacking. The difficulty of accurately measuring NH2OH in water samples severely limits studies. Current spectrophotometric methods have detection limits that are well above concentrations in most natural waters. The most common method to measure NH2OH is oxidation to N2O by ferric ammonium sulfate (FAS) in acidic conditions and measurement of N2O via gas chromatography. While this method has a detection limit that is suitable for measuring NH2OH concentrations in natural waters (< 200 nM), the conversion of NH2OH to N2O is often incomplete, has a significant reaction time (≥ 3 hrs), and requires a recovery curve. Given the reaction rate and efficiency of NH2OH oxidation to N2O by Mn oxides, I present an alternative to this method by using a commercially available Mn oxide, pyrolusite, in place of FAS (Chapter 4). This reaction occurs readily at circumneutral pH and goes to completion in about an hour, eliminating the need for sample acidification, reducing analysis time, and removing the need for a recovery curve. In addition to the three core datasets in this dissertation, I conducted additional related studies on high-resolution profiles of N2O in continental shelf sediments (App. A), abiotic oxidation of NH3 by Mn(III) pyrophosphate (App. B), and NH2OH oxidation by Fe(III) (App. C). In shelf sediments off Cape Hatteras, NC, N2O peaks when O2 is depleted and when Fe2+ concentrations peak, implying the possibility of chemo-denitrification. Abiotic incubations with NH3 and soluble Mn(III) pyrophosphate did not produce N2O, confirming a biotic source of N2O. I measured rates of N2O production by Fe(III) and found the reaction to be very slow compared to oxidation by Mn(III/IV). In summary, this dissertation addresses knowledge gaps in the role that NH2OH and Mn(III/IV)Ox play in marine N2O emissions by providing environmentally relevant rate laws and constants for NH2OH chemo-oxidation and methods to better characterize the biogeochemistry of NH2OH. Using real-time kinetics, cost-effective spatial analysis via microscopy, and a new rapid measurement of NH2OH, this dissertation presents preliminary evidence that NH2OH chemo-oxidation is environmentally relevant in marine systems. Additionally, the methods developed in this dissertation can be used to further build our understanding of N-Mn coupled biogeochemistry in marine systems. Future studies can accurately and rapidly measure marine NH2OH and other reactive intermediates to better constrain the biogeochemical role of coupled biotic-abiotic and N-Mn interactions.
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