Hugh L. MacIntyre

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.

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5‐chloromethylfluorescein diacetate

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5‐chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat‐killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per‐cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live‐dead classification was essentially error free. But overlap between the frequency distributions of living and heat‐killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat‐killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8–10 of 24 species (i.e., 33%–42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone.

The photobiology of Heterosigma akashiwo. Photoacclimation, diurnal periodicity, and its ability to rapidly exploit exposure to high light.

Periodic and seasonal exposure to high light is a common occurrence for many near‐shore and estuarine phytoplankton. Rapid acclimatization to shifts in light may provide an axis by which some species of phytoplankton can outcompete other microalgae. Patterns of photoacclimation and photosynthetic capacity in the raphidophyte Heterosigma akashiwo (Hada) Hada ex Hara et Chihara isolated from the mid‐Atlantic of the United States were followed in continuous cultures at low‐ and high‐light intensities, followed by reciprocal shifts to the opposite light level. The maximum quantum yield (Fv/Fm) as well as the photosynthetic cross‐section (σPSII) of photosystem II was higher in high‐light cultures compared to low‐light cultures. Significant diurnal variability in photochemistry and photoprotection was noted at both light levels, and high‐light‐acclimated cultures displayed greater variability in photoprotective pathways. When shifted from low to high light, there was only a slight and temporary decline in maximum quantum yield, while cell specific growth more than doubled within 24 h. Rapid acclimation to high light was facilitated by short‐term photoprotection (nonphotochemical quenching), reduced PSII reaction center connectivity, and electron transport. Short‐term increases in de‐epoxidated xanthophyll pigments contributed to nonphotochemical protection, but lagged behind initial increases in nonphotochemical quenching and were not the primary pathway of photoprotection in this alga. By 48 h, photochemistry of cultures shifted from low to high light resembled long‐term high‐light‐acclimated cultures. This isolate of H. akashiwo appears well poised to exploit rapid shifts in light by using unique cellular adjustments in light harvesting and photochemistry.

Curvature in models of the photosynthesis-irradiance response

An equation for the rate of photosynthesis as a function of irradiance introduced by T. T. Bannister included an empirical parameter b to account for observed variations in curvature between the initial slope and the maximum rate of photosynthesis. Yet researchers have generally favored equations with fixed curvature, possibly because b was viewed as having no physiological meaning. We developed an analytic photosynthesis‐irradiance equation relating variations in curvature to changes in the degree of connectivity between photosystems, and also considered a recently published alternative, based on changes in the size of the plastoquinone pool. When fitted to a set of 185 observed photosynthesis‐irradiance curves, it was found that the Bannister equation provided the best fit more frequently compared to either of the analytic equations. While Bannisters curvature parameter engendered negligible improvement in the statistical fit to the study data, we argued that the parameter is nevertheless quite useful because it allows for consistent estimates of initial slope and saturation irradiance for observations exhibiting a range of curvatures, which would otherwise have to be fitted to different fixed‐curvature equations. Using theoretical models, we also found that intra‐ and intercellular self‐shading can result in biased estimates of both curvature and the saturation irradiance parameter. We concluded that Bannisters is the best currently available equation accounting for variations in curvature precisely because it does not assign inappropriate physiological meaning to its curvature parameter, and we proposed that b should be thought of as the expression of the integration of all factors impacting curvature.