Type of Document Dissertation Author Gihring, Thomas M. URN etd-05082009-110340 Title Direct Determination of Nitrogen Removal Rates and Pathways in Coastal Ecosystems Degree Doctor of Philosophy Department Oceanography, Department of Advisory Committee
Advisor Name Title Joel Kostka Committee Chair Jeff Chanton Committee Member Markus Huettel Committee Member Stefan Green Committee Member Munir Humayun Outside Committee Member Keywords
Date of Defense 2009-04-27 Availability unrestricted AbstractThis dissertation examines the role of microorganisms in marine biogeochemical cycles with a particular emphasis on sedimentary nitrogen transformations. Nitrogen is required by all living organisms and is a key nutrient controlling the productivity of Earth’s oceans. Pervasive endeavors of modern human society, such as fossil fuel combustion and Haber-Bosch N2 fixation for agricultural fertilizers, have caused large-scale perturbations in the natural, global nitrogen cycle such that the rate of anthropogenic reactive nitrogen creation now exceeds that of all natural processes combined. A substantial fraction of this man-made reactive nitrogen is being lost to the environment where, as a macro-nutrient, excessive nitrogen loading is causing extensive disruptions to natural primary production cycles and food webs. Basic scientific research on nitrogen cycling in coastal oceans is imperative as human activities are increasingly adding to the reactive nitrogen influx to near-shore environments.
Shallow coastal areas (< 200 m water depth) cover only ~ 7% of the ocean although up to 30% of marine primary production occurs in these zones. Biogenic debris settling in coastal zones largely escapes degradation in the water column, and thus, as much as 60% of locally-produced organic matter undergoes benthic deposition and diagenesis. Nutrients regenerated during organic matter mineralization in shallow sediments are essential in fueling high rates of marine primary production in continental margins. Coastal sediments are also important sites of reactive nitrogen removal with the majority of marine microbial N2 production occurring in these areas. Production of N2 in continental shelf sediments via microbial denitrification and anammox (anaerobic ammonium oxidation) is an essential process for nitrogen removal and maintaining a balance of reactive nitrogen in the oceans. Denitrification and anammox are two of the least understood pathways in the nitrogen cycle; explorations of rates and mechanisms for N2 production have been hindered mainly by methodological difficulties, spatial and temporal variability in benthic processes, and previously-overlooked nitrogen cycling pathways. Due largely to a paucity of direct N2 flux rate measurements, most global marine nitrogen budget estimates are tenuous (Capone 2008). Thus, the foci of this dissertation were to better constrain known rates of denitrification and anammox, elucidate the principal controls on these processes in situ, and explore the ecology of microorganisms mediating these reactions in coastal sediments.
Rates and controls of nitrogen removal by microbial N2 production were studied at three field areas in two different estuaries. Field research sites within the Apalachicola National Estuarine Research Reserve included stations adjacent to St. George Island in the Gulf of Mexico (for the investigation of nitrogen cycling in permeable sediments) in addition to stations within an oligohaline marsh near the mouth of the Apalachicola River (for a study of nitrogen removal by coastal wetlands). Benthic microbial nitrogen cycling was also studied in fjords of the Svalbard islands in the Arctic Ocean.
The first of four chapters of this dissertation describes a study identifying the microbial taxa which catalyze phytodetritus degradation and denitrification in permeable coastal sediments of the northeast Gulf of Mexico. Coastal benthic environments typically receive intermittent pulses of organic matter following phytoplankton bloom events and permeable sediments have been demonstrated to rapidly degrade this material. Microorganisms act as the primary agents of benthic organic matter decomposition through the production of extracellular hydrolytic enzymes, fermentation, and terminal carbon mineralization coupled with respiratory processes. Although the role of bacteria in the decomposition of organic matter in marine sediments has long been recognized, the mechanisms regulating organic matter decomposition, and relationships between the phylogeny of benthic microorganisms and changes in biogeochemical and ecological function, are under-explored.
In this study of detritus-degrading microorganisms, stable isotope probing experiments were used to track the assimilation of isotopically-labeled substrate into bacterial DNA and to directly link the taxonomic identification of benthic microorganisms with particulate organic matter degradation and denitrification activity. Phytodetritus deposition events were simulated in the laboratory by the addition of 13C-enriched, heat-killed Spirulina cells to intact sediment core incubations. Immediate increases in O2 consumption, N2 efflux, and dissolved inorganic nitrogen efflux were observed following phytodetritus addition relative to unamended treatments, suggesting that the benthic microbial community was poised to immediately begin oxidizing deposited organic matter. Analyses of 16S rRNA gene sequences amplified from 13C-enriched DNA fractions demonstrated that members of the Gammaproteobacteria (Vibrionales and Alteromonadales), Deltaproteobacteria, Actinobacteria, Verrucomicrobia, and Planctomycetes metabolized the phytodetritus amendment. Terminal restriction length polymorphism analyses showed increases in the relative abundance of Gammaproteobacteria, Planctomycetes, and Bacteroidetes with phytodetritus addition. Alphaproteobacteria were identified as metabolically active denitrifiers by phylogenetic analysis of nitrous oxide reductase gene sequences from 13C-enriched DNA fractions. This study provides the first identification of microorganisms responsible for organic matter degradation in marine sediments by DNA sequence analysis. Microbial assemblages recognized for high molecular weight organic matter oxidation in the marine water column were important in catalyzing these processes in permeable sediments.
Permeable sediments are also the focus of Chapter 2 which describes nitrogen cycling over a one-year period in sublittoral sands from two contrasting sites near St. George Island. Nitrogen stable isotope tracer techniques were used to measure N2 production rates and pathways in sediment cores and slurries. To simulate pore-water advection, which occurs in permeable sands due to interactions between water currents and surface topography, intact sediment cores were perfused with aerated seawater. Pore-water perfusion increased denitrification rates up to 2.5-fold and 15-fold for the Apalachicola Bay and Gulf of Mexico sites, respectively, relative to static cores. Seasonal N2 production rates were highest in spring and fall. Denitrified nitrate was derived almost entirely from benthic nitrification at the Gulf site whereas water column nitrate was more important at the Bay site. Stirred chambers with intact sediment cores were used to determine net fluxes O2, N2, nitrate, and ammonium across the sediment-water interface during varied degrees of continuous pore-water exchange. Rates of N2 efflux were directly correlated with the extent of pore-water flow increasing from 0.13 mmol N m-2 d-1 under diffusion-limited solute transport conditions up to 0.87 mmol N m-2 d-1 with pore water advection. Mineralized nitrogen was completely converted to N2 gas in Gulf of Mexico sediments. These data provide clear evidence that permeable sediments are important in nitrogen removal and N2 production occurs over a continuum of rates dependent on bottom current conditions.
Results from a study of benthic nitrogen cycling in two Arctic fjords are presented in Chapter 3. Intact sediment core incubations were used to quantify net fluxes of dissolved inorganic nitrogen, organic nitrogen, organic carbon, and oxygen at the sediment-water interface. Rates of gross denitrification, anammox, nitrification, dissimilatory nitrate reduction to ammonium, and N2 fixation were quantified using core incubations and slurry experiments. Profiles of dissolved inorganic nitrogen in pore-water, and organic carbon and nitrogen in the solid phase, were also obtained. Net nitrogen losses as N2 ranged from 0.152 to 0.453 mmol N m-2 d-1 and denitrification comprised 2% to 11% of total carbon oxidation. Rates of anammox ranged from 20 to 51 μmol N m-2 d-1 and contributed 5% to 23% of gross N2 generation. Nitrification rates were as high as 0.833 mmol N m-2 d-1 and sediments were a substantial source of nitrate to the water column (0.169 to 0.393 mmol N m-2 d-1 efflux). Uptake of ammonium (0.052 to 0.087 mmol N m-2 d-1), dissolved organic nitrogen (0.291 to 0.486 mmol N m-2 d-1), and dissolved organic carbon (1.31 to 2.50 mmol N m-2 d-1) was observed. Benthic nitrogen fixation was estimated at 0.020 mmol N m-2 d-1 in one of the fjords. Dissimilatory nitrate reduction to ammonium and net N2O production were not detected. This study provides direct evidence that nitrogen loss rates, mainly via denitrification, in Arctic sediments rival those measured in temperate or subtropical environments.
Chapter 4 describes a study sponsored by the NOAA National Estuarine Research Reserve System Graduate Research Fellowship program to investigate nitrogen cycling in the Apalachicola River distributary marsh. Coastal, fringing marshes have been thought to remove dissolved inorganic and particulate nitrogen from external sources via benthic denitrification and burial, respectively, and to export considerable loads of dissolved organic nitrogen (DON) to the greater estuary. There is, however, little data available from tidal freshwater and oligohaline marshes to confirm these hypotheses and benthic nitrogen cycling in the Apalachicola National Estuarine Research Reserve (ANERR) marsh has not been previously studied. This work addressed the hypotheses that the ANERR marshes comprise a significant sink of river-derived dissolved inorganic nitrogen through nitrification-denitrification and burial, and provide a substantial source of nitrogen to the Bay as DON. Denitrification rates were measured between July 2006 and August 2008 using intact sediment core incubations. Rates of net N2 flux ranged from 0.23 to 1.72 mmol N m-2 d-1 with a mean of 0.72 mmol N m-2 d-1 for all sites over the course of the study. Preliminary results indicate that burial of particulate nitrogen (1.46 mmol N m-2 d-1) is a larger annualized loss term than denitrification. A study of tide-driven exchange of nitrogen between marsh creeks and river distributaries showed net uptake of nitrate by marsh sediments and export of DON.
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