In the oligotrophic ocean where inorganic phosphate (P i ) concentrations are low, microorganisms supplement their nutrient requirements with phosphorus (P) extracted from dissolved organic matter (DOM). Most P in DOM is bound as phosphate esters, which are hydrolyzed by phosphoesterases to P i . However, a large fraction of DOM-P occurs as phosphonates, reduced organophosphorus compounds with a C P bond that do not yield P i through simple ester hydrolysis alone. Phosphonates require an additional step that cleaves the C P bond and oxidizes P(III) to P(V) to yield P i . Most phosphonates are metabolized by the C-P lyase pathway, which cleaves C P bonds and oxidizes phosphonates to P i , enabling microbial assimilation. While the activity of common phosphoesterases such as alkaline phosphatase and phosphodiesterase can be measured by a fluorescent assay, a comparable method to assess C-P lyase activity (CLA) in natural water samples does not exist. To address this, we synthesized a dansyl-labeled phosphonate compound, and measured its hydrolysis by C-P lyase using high performance liquid chromatography. We found that laboratory cultures of marine bacteria expressing the C-P lyase pathway are able to hydrolyze the dansyl phosphonate, while bacteria expressing other phosphonate degradation pathways do not. Finally, we performed several field tests of the assay to measure water column profiles of CLA at Sta. ALOHA in the North Pacific Subtropical Gyre. Activity was elevated near the deep chlorophyll maximum suggesting high levels of phosphonate degradation in that region.
<p>A large fraction of marine primary production is directed towards the synthesis of polysaccharides, most of which are rapidly degraded by heterotrophs, including heterotrophic microbes. However, a novel class of polysaccharides characterized by high N-acetyl aminosugar and 6-deoxysugar content, escapes rapid degradation and accumulates as a constituent of marine dissolved organic matter (DOM). These polysaccharides, which comprise ~25% of total dissolved organic carbon, also represent a large reservoir of the potentially bioavailable organic N and P stored in DOM.&#160;&#160; To better understand the accumulation and microbial degradation of DOM polysaccharides we used size-exclusion chromatography and diffusion-ordered NMR spectroscopy to examine the size-distribution and composition of DOM recovered from seawater by ultrafiltration. Our results show that DOM polysaccharides are relatively small, with a molecular weight range of 1.3&#8211;7.7 kD and an average molecular weight of ~6 kD in surface waters decreasing to ~3 kD at 900m. Acid hydrolysis of DOM polysaccharides releases a suite of characteristic neutral sugars (glucose, galactose, mannose, rhamnose, fucose and xylose), but most of the polysaccharide (80-90%) resists hydrolysis and undergoes Maillard-like reactions between amino- and reducing sugars. To circumvent this, we modified our hydrolysis conditions to promote sugar-sugar cleavage. With this approach, we were able to generate a suite of oligosaccharides with molecular weights between 0.3-1.8 kD that carry the same spectral characteristics as DOM polysaccharides. We are using transposon insertion sequencing (Tn-seq) of marine bacteria cultured on these oligosaccharides to identify genes and degradation pathways responsible for DOM polysaccharide degradation.</p> <p>&#160;</p>
Marine dissolved organic matter (DOM) is an actively cycling reservoir of carbon containing thousands of unique compounds. To describe the complex dynamics that govern the biological transformation and decomposition of compounds in this molecular black box, models of DOM reactivity use chemical characteristics, as well as environmental parameters, to describe trends in the turnover time of classes of DOM. In this thesis, I describe two projects that examine hypotheses regarding the turnover of two classes of DOM. In the 1st project, I test the assumption made by the size–reactivity continuum hypothesis that high molecular weight (> 1 kDa) DOM (HMWDOM) represents a diagenetic intermediate between large labile material and small recalcitrant compounds. Size-fractions of HMWDOM were collected using size-exclusion chromatography, and the changes in MW and chemical composition of the fractions were studied using diffusion-ordered spectroscopy. The size fraction carbon isotopic values were correlated with the proportion of humic substances in the fractions. Through linear modeling, the apparent radiocarbon ages of the two major components of HMWDOM were determined to be 1-3 yrs and 2-4 kyrs, respectively. Combined with the measurements of MW distribution this work demonstrates that HMWDOM is composed of two components that have contrasting decomposition pathways in the ocean. HMWDOM cannot be treated as a single DOM pool when incorporated into models of DOM diagenesis. The 2nd project in this dissertation examines the remineralization of phosphonates, compounds with a direct C-P bond, in the lower euphotic zone using a newly developed fluorescent assay, which measures the activity of carbon-phosphorus lyase. C-P lyase activity (CLA) profiles from the North Pacific Subtropical Gyre (NPSG) showed a sharp activity maximum near the deep-chlorophyll maximum (DCM). High-resolution nutrient measurements suggest that this subsurface CLA maximum is the result of a high nitrate flux at the top of the nitracline. The composition of particulate-P through the euphotic zone was also examined. While phosphonates were not detected in suspended particles, a significant amount of aminoethylphosphonate was measured in sinking material, suggesting eukaryotic material may be an important source of phosphonates to the ocean.
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