Growth of the harmful benthic cyanobacterium Microseira wollei is driven by legacy sedimentary phosphorous.

Models for cyanobacterial harmful algae blooms (cHABs) in fresh waters are usually predicated on the relationship between cyanobacterial ecology and dissolved nutrients, particularly phosphorous. Here we show legacy sediment-associated phosphorous as the primary driver of a benthic cHAB, not phosphorous in the water column. Biogeographical surveys by teams of citizen science volunteers working with the University of South Carolina identified over 200 distinct mats of Microseira wollei in Lake Wateree, SC based on toxin characterization. In sum these were estimated to affect approximately 175 km of the lake's shoreline. This growth occurred under water quality conditions that were near or below the regulatory total maximum daily load for phosphorous and nitrogen. A series of established predictive models for cyanobacterial biomass growth were applied retroactively to match the measured growth with measured water quality parameters. The only component of the system that successfully predicted microbial biomass was sedimentary phosphorous. Concentrations of the Lyngbya wollei toxins (LWTs) 1, 4, 5, and 6 were determined at multiple sites over an 18-month period and a toxin inventory for the lake was calculated. Toxin profiles between sites differed at the 95% level of confidence, establishing each site as a unique mat. An empirical model of toxin production potential based on sedimentary phosphorous was developed.

Fe-catalyzed sulfide oxidation in hydrothermal plumes is a source of reactive oxygen species to the ocean.

Historically, the production of reactive oxygen species (ROS) in the ocean has been attributed to photochemical and biochemical reactions. However, hydrothermal vents emit globally significant inventories of reduced Fe and S species that should react rapidly with oxygen in bottom water and serve as a heretofore unmeasured source of ROS. Here, we show that the Fe-catalyzed oxidation of reduced sulfur species in hydrothermal vent plumes in the deep oceans supported the abiotic formation of ROS at concentrations 20 to 100 times higher than the average for photoproduced ROS in surface waters. ROS (measured as hydrogen peroxide) were determined in hydrothermal plumes and seeps during a series of Alvin dives at the North East Pacific Rise. Hydrogen peroxide inventories in emerging plumes were maintained at levels proportional to the oxygen introduced by mixing with bottom water. Fenton chemistry predicts the production of hydroxyl radical under plume conditions through the reaction of hydrogen peroxide with the abundant reduced Fe in hydrothermal plumes. A model of the hydroxyl radical fate under plume conditions supports the role of plume ROS in the alteration of refractory organic molecules in seawater. The ocean's volume circulates through hydrothermal plumes on timescales similar to the age of refractory dissolved organic carbon. Thus, plume-generated ROS can initiate reactions that may affect global ocean carbon inventories.

Asymmetric Versus Symmetric Filter Wheels and Associated Processing Algorithms: Results from Asynchronous Fluorescence Imaging Photometer Measurements of Phytoplankton.

The use of rotating filter wheels is common in photometric applications. Traditional filter wheel designs typically exhibit a number of filter openings spaced evenly about the circumference of the wheel. In this work we examine a number of shortcomings of this traditional filter design in measurements of phytoplankton fluorescence made with our fluorescence imaging photometer (FIP). We present an alternative asymmetric wheel design that offers a number of advantages over the traditional design as well as a new processing algorithm designed to accommodate convolution of signals from adjacent channels inherent in measurements collected with the asymmetric design. This approach eliminates the need for a separate signal to establish timing and wheel position, unambiguously establishes filter order even when the direction of rotation is unknown, allows for better estimates of signal baseline, and is more resilient to effects of vibration and other dynamic processes that could occur on the time scale of wheel rotation. We demonstrate performance improvements for phytoplankton fluorescence measurements associated with the new wheel design and algorithm compared with previously published methods using the FIP. Both the improved image processing algorithm and filter wheel design were found to reduce noise in our measurements significantly.

Single-Cell and Bulk Fluorescence Excitation Signatures of Seven Phytoplankton Species During Nitrogen Depletion and Resupply.

Phytoplankton play a vital role as primary producers in aquatic ecosystems. One common approach to classifying phytoplankton is fluorescence excitation spectroscopy, which leverages the variation in types and concentrations of pigments among different phytoplankton taxonomic groups. Here, we used a fluorescence imaging photometer to measure excitation ratios ("signatures") of single cells and bulk cultures of seven differently pigmented phytoplankton species as they progressed from nitrogen N-replete to N-depleted conditions. Our objective was to determine whether N depletion alters the fluorescence excitation signature of each species and, if so, how quickly they recover when N (as nitrate) was resupplied, because these factors affect our ability to classify the species correctly. Of the seven species studied, only Proteomonas sulcata, a marine cryptophyte, showed measurable changes in single-cell fluorescence excitation ratios and bulk fluorescence excitation spectra. These changes were likely due to decreases in the cellular concentration of phycoerythrin, a N-rich pigment, as N became scarce. Within 3 h of resupply of N, fluorescence signatures began returning to pre-depletion values and were indistinguishable from N-replete cells by 80 h after resupply. These data suggest that our classification approach is robust for non-PE containing phytoplankton. PE-containing phytoplankton might exhibit systematic changes in their signatures depending on their level of N depletion, but this could be detected and the phytoplankton re-classified following a few hours of incubation in N replete conditions.

Fluorescence Excitation Spectroscopy for Phytoplankton Species Classification Using an All-Pairs Method: Characterization of a System with Unexpectedly Low Rank.

An all-pairs method is used to analyze phytoplankton fluorescence excitation spectra. An initial set of nine phytoplankton species is analyzed in pairwise fashion to select two optical filter sets, and then the two filter sets are used to explore variations among a total of 31 species in a single-cell fluorescence imaging photometer. Results are presented in terms of pair analyses; we report that 411 of the 465 possible pairings of the larger group of 31 species can be distinguished using the initial nine-species-based selection of optical filters. A bootstrap analysis based on the larger data set shows that the distribution of possible pair separation results based on a randomly selected nine-species initial calibration set is strongly peaked in the 410-415 pair separation range, consistent with our experimental result. Further, the result for filter selection using all 31 species is also 411 pair separations; The set of phytoplankton fluorescence excitation spectra is intuitively high in rank due to the number and variety of pigments that contribute to the spectrum. However, the results in this report are consistent with an effective rank as determined by a variety of heuristic and statistical methods in the range of 2-3. These results are reviewed in consideration of how consistent the filter selections are from model to model for the data presented here. We discuss the common observation that rank is generally found to be relatively low even in many seemingly complex circumstances, so that it may be productive to assume a low rank from the beginning. If a low-rank hypothesis is valid, then relatively few samples are needed to explore an experimental space. Under very restricted circumstances for uniformly distributed samples, the minimum number for an initial analysis might be as low as 8-11 random samples for 1-3 factors.

Focus-independent particle size measurement from streak images: a comparison of multivariate methods.

Our laboratories have recently developed a flow-through imaging photometer to characterize and classify fluorescent particles between 3 and 47 µm in size. The wide aperture of the objective lens (0.7 NA) required for measuring spectral fluorescence of single particles restricts the depth of field, such that a large sample volume results in many particles that are out of focus. Here, we describe numerical methods for determining the size of these objects, regardless of their distance from the focal plane, using image processing and multivariate calibration. An intensity profile is extracted from the images and is used as the input for a variety of calibration methods, including partial least squares, neural networks, and support vector machines. The capabilities of these methods are examined to establish the best method for particle sizing that is independent of focus. We found that support vector machines provided the best results, with size estimation error of ±3.1 µm.

Geochemical production of reactive oxygen species from biogeochemically reduced Fe.

The photochemical reduction of Fe(III) complexes to Fe(II) is a well-known initiation step for the production of reactive oxygen species (ROS) in sunlit waters. Here we show a geochemical mechanism for the same in dark environments based on the tidally driven, episodic movement of anoxic groundwaters through oxidized, Fe(III) rich sediments. Sediment samples were collected from the top 5 cm of sediment in a saline tidal creek in the estuary at Murrell's Inlet, South Carolina and characterized with respect to total Fe, acid volatile sulfides, and organic carbon content. These sediments were air-dried, resuspended in aerated solution, then exposed to aqueous sulfide at a range of concentrations chosen to replicate the conditions characteristic of a tidal cycle, beginning with low tide. No detectable ROS production occurred from this process in the dark until sulfide was added. Sulfide addition resulted in the rapid production of hydrogen peroxide, with maximum concentrations of 3.85 µM. The mechanism of hydrogen peroxide production was tested using a simplified three factor representation of the system based on hydrogen sulfide, Fe(II) and Fe(III). The resulting predictive model for maximum hydrogen peroxide agreed with measured hydrogen peroxide in field-derived samples at the 95% level of confidence, although with a persistent negative bias suggesting a minor undiscovered peroxide source in sediments.

Surface charge controls the fate of Au nanorods in saline estuaries.

This work reports the distribution of negatively charged, gold core nanoparticles in a model marine estuary as a function of time. A single dose of purified polystyrene sulfonate (PSS)-coated gold nanorods was added to a series of three replicate estuarine mesocosms to emulate an abrupt nanoparticle release event to a tidal creek of a Spartina -dominated estuary. The mesocosms contained several phases that were monitored: seawater, natural sediments, mature cordgrass, juvenile northern quahog clam, mud snails, and grass shrimp. Aqueous nanorod concentrations rose rapidly upon initial dosing and then fell to stable levels over the course of approximately 50 h, after which they remained stable for the remainder of the experiment (41 days total). The concentration of nanorods rose in all other phases during the initial phase of the experiment; however, some organisms demonstrated depuration over extended periods of time (100+ h) before removal from the dosed tanks. Clams and biofilm samples were also removed from the contaminated tanks post-exposure to monitor their depuration in pristine seawater. The highest net uptake of gold (mass normalized) occurred in the biofilm phase during the first 24 h, after which it was stable (to the 95% level of confidence) throughout the remainder of the exposure experiment. The results are compared against a previous study of positively charged nanoparticles of the same size to parameterize the role of surface charge in determining nanoparticle fate in complex aquatic environments.

Taxonomic classification of phytoplankton with multivariate optical computing, part I: design and theoretical performance of multivariate optical elements.

Phytoplankton are single-celled, photosynthetic algae and cyanobacteria found in all aquatic environments. Differential pigmentation between phytoplankton taxa allows use of fluorescence excitation spectroscopy for discrimination and classification. For this work, we applied multivariate optical computing (MOC) to emulate linear discriminant vectors of phytoplankton fluorescence excitation spectra by using a simple filter-fluorometer arrangement. We grew nutrient-replete cultures of three differently pigmented species: the coccolithophore Emiliania huxleyi, the diatom Thalassiosira pseudonana, and the cyanobacterium Synechococcus sp. Linear discriminant analysis (LDA) was used to determine a suitable set of linear discriminant functions for classification of these species over an optimal wavelength range. Multivariate optical elements (MOEs) were then designed to predict the linear discriminant scores for the same calibration spectra. The theoretical performance specifications of these MOEs are described.

Taxonomic classification of phytoplankton with multivariate optical computing, part II: design and experimental protocol of a shipboard fluorescence imaging photometer.

Differential pigmentation between phytoplankton allows use of fluorescence excitation spectroscopy for the discrimination and classification of different taxa. Here, we describe the design and performance of a fluorescence imaging photometer that exploits taxonomic differences for discrimination and classification. The fluorescence imaging photometer works by illuminating individual phytoplankton cells through an asynchronous spinning filter wheel, which produces bar code-like streaks in a fluorescence image. A filter position is covered with an opaque filter to create a reference dark position in the filter wheel rotation that is used to match each fluorescence streak with the corresponding filter. Fluorescence intensities of the imaged streaks are then analyzed for the purpose of spectral analysis, which allows taxonomic classification of the organism that produced the streaks. The theoretical performance and signal-to-noise ratio (SNR) specifications of these MOEs are described in Part I of this series. This report describes optical layout, flow cell design, magnification, depth of field, constraints on filter wheel and flow velocities, procedures for blank subtraction and flat-field correction, the measurement scheme of the instrument, and measurement of SNR as a measurement of filter wheel frequency. This is followed by an analysis of the sources of variance in measurements made by the photometer on the coccolithophore Emiliania huxleyi. We conclude that the SNR of E. huxleyi measurements is not limited by the sensitivity or noise attributes of the measurement system, but by dynamics in the fluorescence efficiency of the E. huxleyi cells. Even so, the minimum SNR requirements given in Part I for the instrument are met.

Taxonomic classification of phytoplankton with multivariate optical computing, part III: demonstration.

We describe the automatic analysis of fluorescence tracks of phytoplankton recorded with a fluorescence imaging photometer. The optical components and construction of the photometer were described in Part I and Part II of this series in this issue. An algorithm first isolates tracks corresponding to a single phytoplankter transit in the nominal focal plane of a flow cell. Then, the fluorescence streaks in the track that correspond to individual optical elements on the filter wheel are identified. The fluorescence intensity of each streak is integrated and used to calculate ratios. This approach was tested using 853 fluorescence measurements of the coccolithophore Emiliania huxleyi and the diatom Thalassiosira pseudonana. Average intensity ratios for the two classes closely follow those predicted in Part I of this series, with a distribution of ratios in each class that is consistent with the signal-to-noise ratio calculations in Part II for single cells. No overlap of the two class ratios was observed, yielding perfect classification.

Combinatorial parameter space as an empirical tool for predicting water chemistry: Fe(II) oxidation across a watershed.

Fe(II) oxidation kinetics in surface waters are a complex function of the concentration of several dissolved species that vary geographically and temporally across watersheds. This work reports an empirical, combinatorial investigation of Fe(II) oxidation that simultaneously evaluated these variations across the pH, Fe(II), PO4³â», Cl⁻, Br(-), CO3²â», and natural organic matter (NOM) axes. The work assayed the effects of independent and dependent variables through application of a novel experimental design that varied Fe(II), PO4³â», Cl⁻, Br⁻, and CO3²â» along the pH axis. Each factor was varied across concentration ranges corresponding to the natural variation between typical fresh and salt water. The system was designed to describe the oxidation of Fe(II) that occurs when Fe(II)-rich groundwaters are mixed rapidly with oxic overlaying waters as a result of tidal movement, bioturbation, dredging, and other mixing/resuspension events. Factors and interfactor interactions were statistically evaluated to determine their importance to Fe(II) oxidation at the 95% level of confidence. Significant factors were retained and used to construct predictive numerical models of Fe(II) oxidation rates. Two models (M1 and M2) were constructed to represent the conditional endmembers of unrestricted Fe cycling (M1) and restricted Fe cycling (due to forced precipitation of Fe(III), M2). The models were challenged to predict net Fe(II) oxidation rates across a watershed (the Congaree/Santee rivers, sampled at ten different locations in South Carolina). The models were generally capable of predicting Fe(II) oxidation rates to within the 95% confidence interval, although M2 consistently overpredicted the rate relative to M1. The minimum initial Fe(II) concentration needed to observe Fe cycling is estimated based on the model output.

Short-term Fe cycling during Fe(II) oxidation: exploring joint oxidation and precipitation with a combinatorial system.

The net oxidation of Fe(II)aq by dioxygen initiates a suite of reactions including the oxidation of multiple Fe(II) complexes, generation of secondary oxidants, Fe(III) reduction, and precipitation of insoluble products. This work reports application of a multifactorial strategy to describe the oxidation of Fe(II) under conditions that correspond to those found where Fe(II)-rich groundwaters mix rapidly with overlying oxygenated waters. Response surfaces were constructed describing the relationship of the net oxidation process with mixtures of the common ligands chloride (Cl-), bromide (Br-), total carbonate (CO3(2-)), Fe(II), and Suwannee River natural organic matter (SRNOM) at pH 8.00. Response surfaces were generated in the presence and absence of added phosphate, representing conditional end members corresponding to geographical locations where Fe(III) precipitation is respectively forced and unconstrained. Comparison of net Fe(II) oxidation rates in the presence and absence of phosphate then enabled resolution of the relative contributions of Fe(II) oxidation and Fe(III) reduction to the overall process. The differences between the two surfaces demonstrated the importance of Fe(II) regeneration on the rapid (min) time scale during net oxidation. The minimum Fe(II) concentration necessary to initiate measurable cycling is reported. The presence of reactive oxygen species was evaluated through the use of probes added to the center point condition of the experimental matrix. Analysis of the statistical significance of the Fe(II)-factor relationships demonstrated that over the conditional scale of the experiments complexation of Fe(II) by the selected ligands did not correlate to the experimental outcome.

Multivariate examination of Fe(II)/Fe(III) cycling and consequent hydroxyl radical generation.

The introduction of Fe(II)(aq) into aerated solutions resulted in net Fe(II) oxidation with concomitant, rapid Fe(II)/Fe(IIII) cycling and concurrent generation of reactive oxygen species. The effect of mixtures of naturally occurring solutes on Fe(II)/Fe(III) cycling and the concurrent oxidation of dissolved organics is reported. The experimental strategy was based on a multivariate, microscale, high-throughput approach for evaluating the effect of covarying concentrations of bromide, iodide, Suwannee River natural organic matter (SRNOM), chloride, and total carbonate species. Superoxide and HO• were evaluated at the center point condition of the experimental design with selective scavengers (superoxide dismutase and benzoic acid). The rate of Fe(II) oxidation decreased in the presence of these scavengers, indicating it is a function of oxygen, superoxide, and HO•. HO• generated during Fe(II)/Fe(III) cycling was quantified with the selective probe 1,3-dicyanotetrachlorobenzene (DCTCB). Through the range of experimental conditions of this design, the ratio of the number of moles of HO• produced to the number of moles of Fe(II) consumed varied from 3 to 750, corresponding to approximately 10 to 2200 Fe(II)/Fe(III) cycles, respectively. The implications of these findings with respect to organic oxidation during the aeration of anoxic Fe(II) rich groundwaters are discussed.

Construction, figures of merit, and testing of a single-cell fluorescence excitation spectroscopy system.

Characterization of phytoplankton community composition is critical to understanding the ecology and biogeochemistry of the oceans. One approach to taxonomic characterization takes advantage of differing pigmentation between algal taxa and thus differences in fluorescence excitation spectra. Analyses of bulk water samples, however, may be confounded by interference from chromophoric dissolved organic matter or suspended particulate matter. Here, we describe an instrument that uses a laser trap based on a Nikon TE2000-U microscope to position individual phytoplankton cells for confocal fluorescence excitation spectroscopy, thus avoiding interference from the surrounding medium. Quantitative measurements of optical power give data in the form of photons emitted per photon of exposure for an individual phytoplankton cell. Residence times for individual phytoplankton in the instrument can be as long as several minutes with no substantial change in their fluorescence excitation spectra. The laser trap was found to generate two-photon fluorescence from the organisms so a modification was made to release the trap momentarily during data acquisition. Typical signal levels for an individual cell are in the range of 10(6) photons/s of fluorescence using a monochromated 75 W Xe arc lamp excitation source with a 2% transmission neutral density filter.

Transfer of gold nanoparticles from the water column to the estuarine food web.

Within the next five years the manufacture of large quantities of nanomaterials may lead to unintended contamination of terrestrial and aquatic ecosystems. The unique physical, chemical and electronic properties of nanomaterials allow new modes of interaction with environmental systems that can have unexpected impacts. Here, we show that gold nanorods can readily pass from the water column to the marine food web in three laboratory-constructed estuarine mesocosms containing sea water, sediment, sea grass, microbes, biofilms, snails, clams, shrimp and fish. A single dose of gold nanorods (65 nm length x 15 nm diameter) was added to each mesocosm and their distribution in the aqueous and sediment phases monitored over 12 days. Nanorods partitioned between biofilms, sediments, plants, animals and sea water with a recovery of 84.4%. Clams and biofilms accumulated the most nanoparticles on a per mass basis, suggesting that gold nanorods can readily pass from the water column to the marine food web.

Use of multiparametric techniques to quantify the effects of naturally occurring ligands on the kinetics of Fe(II) oxidation.

A multifactorial experimental design was employed to quantify and rank the effects of a series of ligands on the rate of Fe(II) (18 microM) oxidation in a system containing chloride, sulfate, carbonate/bicarbonate, fluoride, and natural organic matter (NOM) at pH 8.34 +/- 0.13. Several factors and combinations thereof correlated with the rate of Fe(II) oxidation at the 95% level of confidence. Presented in decreasing order of significance, those factors were carbonate/bicarbonate, NOM, sulfate, chloride, the sulfate/fluoride interaction, and fluoride. The center point of the experimental design was repeated with different organic matters substituted, including Nordic Reservoir NOM, fulvic and humic acids; Suwannee River NOM, fulvic and humic acids; and Pony Lake fulvic acid. Despite the widely differing geographical origins of these organic materials, their overall impact on the oxidation rate of Fe(II) was consistent with the observed rate varying no more than a factor of 2 as a function of different organic matters (on a milligrams of carbon per liter basis). The utility of the pentafactorial response surface model (based on Nordic Lake NOM) to predict Fe(II) oxidation rates was evaluated for different natural water samples, including two seawater and one freshwater.

Cellular uptake and cytotoxicity of gold nanorods: molecular origin of cytotoxicity and surface effects.

Gold nanorods of different aspect ratios are prepared using the growth-directing surfactant, cetyltrimethylammonium bromide (CTAB), which forms a bilayer on the gold nanorod surface. Toxicological assays of CTAB-capped nanorod solutions with human colon carcinoma cells (HT-29) reveal that the apparent cytotoxicity is caused by free CTAB in solution. Overcoating the nanorods with polymers substantially reduces cytotoxicity. The number of nanorods taken up per cell, for the different surface coatings, is quantitated by inductively coupled plasma mass spectrometry on washed cells; the number of nanorods per cell varies from 50 to 2300, depending on the surface chemistry. Serum proteins from the biological media, most likely bovine serum albumin, adsorb to gold nanorods, leading to all nanorod samples bearing the same effective charge, regardless of the initial nanorod surface charge. The results suggest that physiochemical surface properties of nanomaterials change substantially after coming into contact with biological media. Such changes should be taken into consideration when examining the biological properties or environmental impact of nanoparticles.

Complexity of neural mechanisms underlying overconsumption of sugar in scheduled feeding: involvement of opioids, orexin, oxytocin and NPY.

A regular daily meal regimen, as opposed to ad libitum consumption, enforces eating at a predefined time and within a short timeframe. Hence, it is important to study food intake regulation in animal feeding models that somewhat reflect this pattern. We investigated the effect of scheduled feeding on the intake of a palatable, high-sugar diet in rats and attempted to define central mechanisms - especially those related to opioid signaling--responsible for overeating sweet foods under such conditions. We found that scheduled access to food, even as challenging as 20 min per day, does not prevent overconsumption of a high-sucrose diet compared to a standard one. An opioid receptor antagonist, naloxone, at 0.3-1 mg/kg b. wt., decreased the intake of the sweet diet, whereas higher doses were required to reduce bland food consumption. Real-time PCR analysis revealed that expression of hypothalamic and brainstem genes encoding opioid peptides and receptors did not differ in sucrose versus regular diet-fed rats, which suggests that scheduled intake of sweet food produces only a transient change in the opioid tone. Intake of sugar was also associated with upregulation of orexin and oxytocin genes in the hypothalamus and NPY in the brainstem. We conclude that scheduled consumption of sugar diets is associated with activity of a complex network of neuroregulators involving opioids, orexin, oxytocin and NPY.

Free-drifting icebergs: hot spots of chemical and biological enrichment in the Weddell Sea.

The proliferation of icebergs from Antarctica over the past decade has raised questions about their potential impact on the surrounding pelagic ecosystem. Two free-drifting icebergs, 0.1 and 30.8 square kilometers in aerial surface area, and the surrounding waters were sampled in the northwest Weddell Sea during austral spring 2005. There was substantial enrichment of terrigenous material, and there were high concentrations of chlorophyll, krill, and seabirds surrounding each iceberg, extending out to a radial distance of approximately 3.7 kilometers. Extrapolating these results to all icebergs in the same size range, with the use of iceberg population estimates from satellite surveys, indicates that they similarly affect 39% of the surface ocean in this region. These results suggest that free-drifting icebergs can substantially affect the pelagic ecosystem of the Southern Ocean and can serve as areas of enhanced production and sequestration of organic carbon to the deep sea.