NER: Exploratory Research on Developing a Nanoscale Sensing Device for Measuring the Supply of Iron to Eukaryotic Phytoplankton in Natural Seawater

The long delay in recognizing the potentially key role of Fe in coastal marine systems has been in large part because of the complexity of microbial:Fe interactions in seawater. There still is no analytical method for determining biologically available Fe for either prokaryotic or eukaryotic phytoplankton. However, there is evidence that Fe availability to eukaryotic phytoplankton can be regulated by additions of the fungal siderophore desferrioxamine B (DFB) to coastal waters. The DFB-Fe complex not only is unavailable for uptake at significant rates, but also outcompetes the natural organic ligand classes in seawater for Fe. Measurement of DFB-Fe concentrations in a titration series should therefore provide a first order measure of the Fe supply to phytoplankton.This project will investigate the feasibility of miniaturizing a current bulk liquid membrane system that can actively isolate and concentrate 59Fe-DFB from aqueous solutions by constructing liposomes with the needed transport characteristics. The high surface area and optimal diffusional aspects of these nanodevices will enable efficient 59Fe-DFB accumulations at the extremely low dissolved Fe concentrations (\u3c1 nM) encountered in coastal waters. The transport characteristics of these nanodevices will be measured as a function of liposome compositions and fabrication conditions to optimize the active transport of 59Fe-DFB from a seawater matrix. If progress permits, the relationship between 59Fe accumulated by these nanodevices and cellular 59Fe uptake and growth of natural population cultures will be explored. The immediate goal of this exploratory project is to demonstrate the feasibility of developing nanoscale sensors for quantifying the supply of Fe to eukaryotic phytoplankton in coastal seawaters. The broader goal is to establish the viability of nanotechnology for sensing bioactive substrates in the marine realm

U.S.-Japan-Hong Kong Planning Visit: Long Term Collaborative Research Studying Fe Effects on Ecosystem Structure in the Subarctic Pacific

This award supports a short-term U.S-Japan-Hong Kong Planning Visit in preparation for a long-term collaborative research project studying Fe effects on ecosystem structure in the Sub arctic Pacific. The collaborators are Professor Mark Wells at the University of Maine and Professor Shigenobu Takeda at the University of Tokyo in Japan and Professor Paul Harrison at the Hong Kong University of Science and Technology. Virtually the entire Sub arctic Pacific to the Aleutian Islands is a High Nitrate Low Chlorophyll (HNLC) region, characterized by persistently elevated concentrations of macronutrients throughout the year. Independent studies have demonstrated that a shortage of the micronutrient iron is responsible for this condition. This finding is important because by controlling the export of carbon from surface waters, iron may influence atmospheric carbon dioxide concentrations and thus global climate. The researchers hope to plan a series of measurements that would be extremely important for understanding the longer term and broader effect of the iron enrichment. The measurements may also answer questions associated with the long-term response of the plankton community, changes in the iron chemistry and the fate of the carbon produced by the Fe enrichment. The collaborators have complementary scientific expertise in the field. Understanding the effects of iron inputs (natural or globally engineered) on ecosystem structure and carbon export in these waters is essential to obtain a predictive understanding of how changes in iron inputs may affect global climate. The exchange of ideas and data with Japanese and Hong Kong experts in the field will enable U.S. participants to advance their own work, and will set the stage for future international collaborative projects

Collaborative Research: What Limits Denitrification and Bacterial Growth in Lake Bonney, Taylor Valley, Antarctica?

Denitrification is the main process by which fixed nitrogen is lost from ecosystems and the regulation of this process may directly affect primary production and carbon cycling over short and long time scales. Previous investigations of the role of bioactive metals in regulating denitrification in bacteria from permanently ice-covered Lake Bonney in the Taylor Valley of East Antarctica indicated that denitrifying bacteria can be negatively affected by metals such as copper, iron, cadmium, lead, chromium, nickel, silver and zinc; and that there is a distinct difference in denitrifying activity between the east and west lobes of the lake. Low iron concentrations were found to exacerbate the potential toxicity of the other metals, while silver has the potential to specifically inhibit denitrification because of its ability to interfere with copper binding in redox proteins, such as nitrite reductase and nitrous oxide reductase. High silver concentrations might prevent the functioning of nitrous oxide reductase in the same way that simple copper limitation does, thereby causing the buildup of nitrous oxide and resulting in a nonfunctional nitrogen cycle. Other factors, such as oxygen concentration, are likely also to affect bacterial activity in Lake Bonney. This project will investigate silver toxicity, general metal toxicity and oxygen concentration to determine their effect on denitrification in the lake by using a suite of sentinel strains of denitrifying bacteria (isolated from the lake) incubated in Lake Bonney water and subjected to various treatments. The physiological responses of these strains to changes in metal and oxygen concentration will be quantified by flow cytometric detection of single cell molecular probes whose sensitivity and interpretation have been optimized for the sentinel strains. Understanding the relationships between metals and denitrification is expected to enhance our understanding of not only Lake Bonney\u27s unusual nitrogen cycle, but more generally, of the potential role of metals in the regulation of microbial nitrogen transformations.The broader impacts of this work include not only a better understanding of regional biogeochemistry and global perspectives on these processes; but also the training of graduate students and a substantial outreach effort for school children

RAPID: A Unique Cruise Opportunity to Test the Effect of Trace Metal Limitation on Oxidative Stress and Coral Bleaching

Intellectual Merit. Coral bleaching has increased dramatically in frequency, severity, and geographic extent since the 1980\u27s and this trend is anticipated to continue, causing major environmental and economic impacts in tropical regions. This bleaching, or loss by corals of their photosynthetic endosymbiotic dinoflagellates (zooxanthellae; Symbiodinium spp.), appears to result from increased oxidative stress arising from the combined effects of elevated temperature at high light intensities. However, the mechanisms underlying this failure are not understood. The premise of the PIs\u27 current project entitled Effects of Trace Metal Limitation on Oxidative Stress in Zooxanthellae and Its Role in Coral Bleaching (OCE - 0648478) is that the necessary up-regulation of zooxanthellae antioxidant defenses is restricted by low concentrations of dissolved Fe, Zn and Cu; metals essential for antioxidant enzyme function (Cu, Zn-, Mn-, and Fe-SOD; catalase [Fe]; ascorbate peroxidase [Fe]). Findings from their laboratory and field manipulation experiments show that restricting Fe, and Cu/Zn availability to coral hosts under high (but not low) temperature and light intensity indeed can significantly decrease both photosynthetic efficiency of symbionts in-hospite and ROS enzyme activities, while increasing non-photosynthetic quenching of their photosystems; each indicators of the onset of bleaching conditions. However, although there is high integrity within each experiment, they have found this pattern is not consistent with all coral colonies. Based on limited sampling, it appears that corals collected from the outer shelf region normally (but not always) display indications of oxidative stress under conditions of decreased metal availability, while those collected nearshore, or maintained in coastally-derived flowing seawater (where dissolved metal concentrations are higher), often show little discernable effect. Excess metal uptake and storage is well described in the marine phytoplankton literature, which suggests that the history of the coral metal exposure is a critical factor, both with respect to our experiments as well as to the distribution of coral bleaching observed. The PIs have an unique and unforeseen opportunity to test this hypothesis by joining an Australian Institute for Marine Sciences research cruise to Flinders Reef; an offshore atoll in the Coral Sea that is substantially more distant from sporadic terrestrial metal inputs that our previous study sites. They will participate in this cruise to run on-deck incubations testing the effect of reduced and marginally elevated Fe, Cu, Zn and Mn concentrations on coral photosynthetic efficiency, ROS enzyme activities, symbiont pigment composition, and ROS enzyme and other gene expression. This geographical site will provide the ideal test site for verifying their findings of metal effects on oxidative stress in zooxanthellae, and identify some of the key mechanisms and nutritional factors contributing to the increasingly frequent and severe coral bleaching events in tropical waters.Broader Impacts: This project will provide a unique research opportunity for two graduate students and a junior female Ph.D. scientist, who will use aspects of the work for their thesis and career development. The research addresses the fundamental unknowns of the controls of coral bleaching, one of the leading threats to marine biodiversity and economic stability of tropical nations. The findings will provide a key test of laboratory- and field-developed hypotheses of the role of trace metal limitation as a contributor to oxidative stress of zooxanthellae and their coral hosts; a precursor to coral bleaching. A modular series of lectures and demonstrations targeting both upper K-12 and undergraduates will be developed and will be incorporated into existing outreach programs and undergraduate courses in Marine Science at the University of Maine. The phototrophic symbiosis between zooxanthellae and corals, and its disruption by physical environmental factors, provides an inherently powerful case study for the integration of chemistry, physics, and biology that will illustrate to marine science undergraduates the need for rigorous training in the quantitative physical sciences. The findings will provide key insights to the factors that influence the severity of bleaching events, and possibly suggest realistic mitigation strategies to minimize bleaching events in localized environmentally or economically sensitive regions

The Effect of Rate and Source of Potassium Fertilizer on Cured Leaf Yield of Burley Tobacco and Leaf Content and Soil Test Levels of Potassium and Magnesium

In response to questions being asked by tobacco growers about the effectiveness of sulfate of potash magnesia (SPM; 21% K2O and 11% Mg) as coinpared to sulfate of potash (SP; 50% K2O), field studies were conducted during 1993-1994 to compare the two potassium (K) sources for use on burley tobacco. Any effect of SPM on yield of tobacco should be due to Mg since the only difference between the two sources in kind of nutrient contained is the presence of magnesium (Mg) in SPM. To compare the two K sources, we selected field sites low enough in soil test K levels that normally would result in increased tobacco yields due to application of fertilizer K

Collaborative Research: The Effect of Iron-Complexing Ligands on Iron Availability to Phytoplankton in HNLC Waters of the Subarctic Pacific Ocean

Scientists from the University of Maine and San Francisco State University propose to do deck-board incubation experiments in high nutrient, low chlorophyll (HNLC) waters of the eastern (Ocean Station PAPA) and the western (Ocean Station KNOT) Subarctic Pacific Ocean to determine how Fe supply affects phytoplankton species composition. Specifically, this team of scientists plans to address the following specific objectives: (1) assess how the relative availability of Fe bound to weaker and stronger classes of ligands differs among different phytoplankton groups (cyanobacteria, diatoms, dinoflagellates, prymnesiophytes) and how these differences influence the evolution of the phytoplankton community after Fe enrichment in HNLC waters; (2) ascertain if new ligands produced in response to Fe enrichment of HNLC waters behave similarly to ambient ligands, or if they have significantly different effect on regulating how an ecosystem evolves over the long term; and (3) determine whether phytoplankton assemblages in HNLC waters having different proximity or history of Fe inputs respond differently to the same suite of Fe ligand blends, or whether conditioning has led to their adaptation of alternate uptake capabilities. In addition, measurements of growth rates, macronutrient utilization rates, fluorescence, cell size determinations, Fe use efficiencies, rates of Fe and carbon uptake and flow cytometry sorting will be done to assess how specific organisms will respond to Fe supplied in different chemical forms

The role of colloid chemistry in providing a source of iron to phytoplankton

Culture experiments with the coastal marine diatom Thalassiosira pseudonana (WHOI clone 3H) demonstrate that, as an iron source, freshly prepared colloidal ferric hydroxide can produce better cell yield than the more crystalline goethite or hematite, Ageing or heating of the prepared ferric hydroxide stock causes a reduction in cell yield. This reduction appears to be related to increased thermodynamic stability of the colloid as suggested by thermogravimetric analysis and relative dissolution rates. The reduction in cell yield can be prevented by the addition of the chelating agent EDTA prior to, but not after, ageing or heating of the ferric hydroxide stock. These results suggest that the ability of colloidal iron to provide a source of metal for phytoplankton is related to the thermodynamic stability of the colloid

NIRT: Developing a Nanoscale Sensing Device for Measuring the Supply of Iron to Phytoplankton in Marine Systems

There is increasing evidence that Fe has a singularly unique role in marine ecosystems, both regulating total phytoplankton production in high nitrate, low chlorophyll regions of the world, and influencing the predominant composition of the phytoplankton assemblages found in others. It is remarkable then that there is no agreement about how to define biologically available Fe, in contrast to the macronutrients nitrogen, phosphorous or silicon. Current attempts to attain predictive insights to how ocean ecosystems will influence the magnitude of climate change are blocked in large part by this question, along with an extreme shortage of data on Fe distributions in the oceans. There is recent evidence that Fe availability can be regulated in bulk seawater incubations by small additions of the fungal siderophore desferrioximine B (DFB). The Fe-DFB complex is not readily available to eukaryotic phytoplankton, so that if the quantity of Fe complexed by DFB were measured and calibrated to Fe uptake by phytoplankton it could yield a novel first order measure of Fe availability. Building from our current research we have developed liposomes that specifically acquire DFB-bound Fe from solution. These devices, 100 nm in diameter, open the way to applying nanotechnology to create a new breed of Fe biosensors in marine waters. The project goals are to 1) optimize these nanodevices by improving their physical robustness, identifying the size/functionality relationship, and examining the efficacy of other DFB-Fe transporter molecules, 2) develop self-reporting capabilities for quantifying Fe uptake by these nanodevices, and 3) to calibrate the capture of Fe by these nanodevices to the Fe uptake by various phytoplankton species. The anticipated final product will be a calibrated nanoscale biosensor for laboratory-scale use that could then be adapted for deploying on remote vehicles. Broader Impacts Resulting from the Proposed Activity: The two institutions involved in this project (U. Maine and Colby College) have a strong track record for involving undergraduate and graduate students in cutting edge research in marine science and chemistry, and this project will continue this process

Characterization of Putative Iron Responsive Genes as Species-Specific Indicators of Iron Stress in Thalassiosiroid Diatoms

Iron (Fe) availability restricts diatom growth and primary production in large areas of the oceans. It is a challenge to assess the bulk Fe nutritional health of natural diatom populations, since species can differ in their physiological and molecular responses to Fe limitation. We assayed expression of selected genes in diatoms from the Thalassiosira genus to assess their potential utility as species-specific molecular markers to indicate Fe status in natural diatom assemblages. In this study, we compared the expression of the photosynthetic genes encoding ferredoxin (a Fe-requiring protein) and flavodoxin (a Fe-free protein) in culture experiments with Fe replete and Fe stressed Thalassiosira pseudonana (CCMP 1335) isolated from coastal waters and Thalassiosira weissflogii (CCMP 1010) isolated from the open ocean. In T. pseudonana, expression of flavodoxin and ferredoxin genes were not sensitive to Fe status but were found to display diel periodicities. In T. weissflogii, expression of flavodoxin was highly responsive to iron levels and was only detectable when cultures were Fe limited. Flavodoxin genes have been duplicated in most diatoms with available genome data and we show that T. pseudonana has lost its copy related to the Fe-responsive copy in T. weissflogii. We also examined the expression of genes for a putative high affinity, copper (Cu)-dependent Fe uptake system in T. pseudonana. Our results indicate that genes encoding putative Cu transporters, a multi-Cu oxidase, and a Fe reductase are not linked to Fe status. The expression of a second putative Fe reductase increased in Fe limited cultures, but this gene was also highly expressed in Fe replete cultures, indicating it may not be a useful marker in the field. Our findings highlight that Fe metabolism may differ among diatoms even within a genus and show a need to validate responses in different species as part of the development pipeline for genetic markers of Fe status in field populations