Tammi L. Richardson

Spectral fluorometric characterization of phytoplankton community composition using the Algae Online Analyser

The utility of a multiple-fixed-wavelength spectral fluorometer, the Algae Online Analyser (AOA), as a means of quantifying phytoplankton biomass and community composition was tested using natural communities from two southeastern United States estuaries, North Inlet, South Carolina, and the Neuse River Estuary, North Carolina. Estimates of biomass (as chlorophyll a) were correlated with HPLC values and variations (usually over-estimates) were consistent with effects of light intensity and nutrient availability on fluorescence quenching. AOA estimates of taxonomic structure were consistent with those from HPLC-derived marker pigments by ChemTax, with both methods indicating domination by chromophytes and green algae in North Inlet and chromophytes and cyanobacteria in the Neuse. We recommend frequent calibration by discrete sample collection, and calibration with species representative of the region of interest. Overall, the AOA appears to be a useful tool for monitoring of phytoplankton community composition, especially as an early warning system for the detection of harmful algal blooms.

Taxonomic Discrimination of Phytoplankton by Spectral Fluorescence

Chlorophyll fluorescence techniques are used widely in both laboratory and field studies to assess the abundance and physiological responses of cyanobacteria, microalgae, macroalgae and vascular plants, as described in other chapters in this volume. Most of the instruments used in these studies excite fluorescence in the blue region of the spectrum and measure chlorophyll fluorescence (peak ca. 685 nm) at ambient temperature. Fluorescence is generally detected using a photomultiplier tube (PMT), which is very sensitive to intensity but insensitive to spectral quality. Cross-talk between the light source used to excite fluorescence and the detector is prevented by the use of cut-off filters on both the emitter and the PMT, or by the use of emitters with narrow wavebands, such as light-emitting diodes (LEDs) or lasers, and a long-pass filter on the detector. With the advent of LEDs, which have a very high efficiency (intensity of light output per unit power input) compared to the xenon flash-lamps used in many older instruments, commercially-available fluorometers can have very low power demands and be both small and sensitive (detection limits are typically <1 mg m−3 of Chla). This makes them ideal for unattended monitoring such as on platforms, moorings or gliders.

Monitoring of the toxic dinoflagellate Karenia brevis using gyroxanthin-based detection methods

The threat to human health and fisheries resources due to blooms of the toxic dinoflagellate Karenia brevis has lead to widespread public concern and calls for continuous monitoring of coastal waters for this organism. Here, a rapid and sensitive photopigment-based monitoring approach is described that incorporates refinements to standard filtration and analytical methods. This method uses the biomarker pigment gyroxanthin-diester contained in cells of some gymnodiniod species including K. brevis. Investigations of the retention efficiencies of five filter types for gyroxanthin from natural blooms of K. brevis showed no significant differences between GF/F, GF/C, 934-AH, GF/A or GF/D filters. Retention efficiencies were generally greater than 98% of cells added, indicating that the larger nominal pore size filters may be used safely for sample collection, reducing overall filtration times for large volumes of water. Simulated bloom experiments using cultures of K. brevis added to unfiltered water from Galveston Bay showed that retention of gyroxanthin on GF/D filters was significantly lower than on other filter types. There were significant interactions (p < 0.01) between filter type and cell density for the variables gyroxanthin, gyroxanthin chl a−1 and gyroxanthin cell−1, suggesting that the performance of the different filter types was dependent on cell density. Retention efficiencies for the simulated blooms ranged between >99% of cells retained and <30% of cells retained (greatest losses were for the GF/D filters). Combined results of natural and simulated blooms indicated that GF/C, 934-AH or GF/A filters gave the best retention efficiency with the fastest filtration times. Sample processing times were also improved by modifying the flow gradients in an existing HPLC protocol allowing the analysis of 106 samples in 24 h. The resulting protocol is suitable for incorporation into routine water quality monitoring programs, and would greatly facilitate the early detection and tracking of K. brevis blooms in coastal waters.

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.

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.

Linear Discriminant Analysis of Single-Cell Fluorescence Excitation Spectra of Five Phytoplankton Species

Linear discriminant analysis (LDA) of single-cell fluorescence excitation spectra (λem=680 nm) for five species of marine phytoplankton was used to determine whether intra-species variation among single cells precluded discrimination among species. Single-cell spectra were recorded in an optical trap with a custom-built spectral fluorometer. For nitrogen (N)- replete cells, separation of all five species (Emiliania huxleyi, a coccolithophore, Thalassiosira pseudonana, a diatom, Dunaliella tertiolecta, a chlorophyte, Amphidinium carterae, a dinoflagellate, and Rhodomonas salina, a cryptophyte) was possible using only a portion of the excitation spectra (570–610 nm). This wavelength region gave perfect classification of species with a minimum Fisher ratio of 62. For four species (E. huxleyi, T. pseudonana, D. tertiolecta, and A. carterae), variations in fluorescence excitation spectra as cells were starved of N did not impact the classification process adversely within the chosen spectral window. R. salina cells grown with and without N showed significant differences in their fluorescence excitation spectra but could still be classified if a different spectral window (490–570 nm) was used. Overall, we conclude that intra-species variation among single-cell fluorescence excitation spectra does not preclude discrimination among species.

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.

Photopigment radiolabelling as a tool for determining in situ growth rates of the toxic dinoflagellate Karenia brevis (Dinophyceae)

Determination of growth rates of harmful algae is critical to understanding bloom dynamics. However, we have few reliable methods of directly determining in situ growth rates on natural populations. One available method is photopigment radiolabelling, where C-specific growth rates are based on synthesis rates of chlorophylls and carotenoids using 14C-bicarbonate as a tracer. Here, we examined radiolabelling of the biomarker pigment gyroxanthin-diester as a tool for determining in situ growth rates of the toxic dinoflagellate Karenia brevis. We also characterized growth responses of K. brevis to various nitrogenous (N) nutrient sources and the presence or absence of a natural plankton community (including grazers) at two irradiances. We found that gyroxanthin radiolabelling may be used successfully to determine C-specific growth rates of K. brevis during monospecific blooms. However, the approach may be of limited use in mixed assemblages when there is low Karenia biomass because low concentrations of gyroxanthin per cell and low turnover rates result in poor signal for the gyroxanthin pigment. Growth rates of K. brevis generally ranged between 0.1 and 0.4 d−1. Cells in batch culture grew equally well on inorganic and organic forms of N, so they may be physiologically capable of using the major N forms present in agricultural run-off, atmospheric deposition, and other anthropogenic inputs. This has implications for the control of K. brevis blooms in nutrient-impacted coastal waters in that organic N inputs should be considered (along with inorganic N) in nutrient management strategies. Our highest C-specific growth rates (0.7 d−1) were measured in the presence of microzooplankton grazers, also indicating that the release of dissolved compounds may play a role in stimulating the growth of K. brevis in nature.

Pigment composition and photoacclimation as keys to the ecological success of Gonyostomum semen (Raphidophyceae, Stramenopiles)

Aquatic habitats are usually structured by light attenuation with depth resulting in different microalgal communities, each one adapted to a certain light regime by their specific pigment composition. Several taxa contain pigments restricted to one phylogenetic group, making them useful as marker pigments in phytoplankton community studies. The nuisance and invasive freshwater microalga Gonyostomum semen (Raphidophyceae) is mainly found in brown water lakes with sharp vertical gradients in light intensity and color. However, its pigment composition and potential photoadaptations have not been comprehensively studied. We analyzed the photopigment composition of 12 genetically different strains of G. semen by high performance liquid chromatography after acclimation to different light conditions. We confirmed the pigments chl a, chl c1c2, diadinoxanthin, trans‐neoxanthin, cis‐neoxanthin, α and β carotene, which have already been reported for G. semen. In addition, we identified, for the first time, the pigments violaxan‐thin, zeaxanthin, and alloxanthin in this species. Alloxanthin has never been observed in raphidophytes before, suggesting differences in evolutionary plastid acquisition between freshwater lineages and the well‐described marine species. The amount of total chl a per cell generally decreased with increasing light intensity. In contrast, the increasing ratios of the prominent pigments diadinoxanthin and alloxanthin per chl a with light intensity suggest photoprotective functions. In addition, we found significant variation in cell‐specific pigment concentration among strains, grouped by lake of origin, which might correspond to genetic differences between strains and populations.