Analysis of spectral excitation for measurements of fluorescence constituents in natural waters

Field measurements of chlorophyll-a (Chl), phycoerythrin (PE), chromophoric dissolved organic matter (CDOM), and variable fluorescence (Fv/Fm) in diverse waters of the California Current, Mediterranean Sea and Gulf of Mexico using 375, 405, 510 and 532 nm laser excitation wavelengths (EW) are analyzed. EW = 375 and 405 nm were found more suitable for Chl assessment in high-Chl (> 10 μg/l) waters. Both EW = 532 and 510 nm can be used to efficiently stimulate PE fluorescence for structural characterization of phytoplankton communities. EW = 375 nm and 405 nm can provide best results for CDOM assessments in offshore oceanic waters; the green EWs can be also used for CDOM measurements in fresh and estuarine water types in conjunction with spectral discrimination between CDOM and PE fluorescence. Both EW = 405 and 510 are suitable for photo-physiological Fv/Fm assessments, though using EW = 405 nm may result in underestimation of PE-containing phytoplankton groups present in mixed phytoplankton assemblages

Advanced laser fluorometry of natural aquatic environments

The Advanced Laser Fluorometer (ALF) provides spectral deconvolution (SDC) analysis of the laser-stimulated emission (LSE) excited at 405 or 532 nm for assessment of chlorophyll a, phycoerythrin, and chromophoric dissolved organic matter. Three spectral types of phycoerythrin are discriminated for characterization of cyanobacteria and cryptophytes in mixed phototrophic populations. The SDC analysis is integrated with measurements of variable fluorescence, Fv/Fm, corrected for the SDC-retrieved background fluorescence, BNC, for improved photophysiological assessments of phytoplankton. The ALF deployments in the Atlantic and Pacific Oceans, and Chesapeake, Delaware, and Monterey Bays revealed significant spectral complexity of LSE. Considerable variability in chlorophyll a fluorescence peak, 673-685 nm, was detected. High correlation (R2 = 0.93) was observed in diverse water types between chlorophyll a concentration and fluorescence normalized to water Raman scattering. Three unidentified red bands, peaking at 625, 644, and 662 nm, were detected in the LSE excited at 405 nm. Significant variability in the BNC/chlorophyll a ratio was observed in diverse waters. Examples of the ALF spectral correction of Fv/Fm, underway shipboard measurements of horizontal variability, and vertical distributions compiled from the discrete samples analyses are presented. The field deployments have demonstrated the utility of the ALF technique as an integrated tool for research and observations

Next generation Advanced Laser Fluorometry (ALF) for characterization of natural aquatic environments: new instruments

The new optical design allows single- or multi-wavelength excitation of laser-stimulated emission (LSE), provides optimized LSE optical collection for spectral and temporal analyses, and incorporates swappable modules for flow-through and small-volume sample measurements. The basic instrument configuration uses 510 nm laser excitation for assessments of chlorophyll-a, phycobiliprotein pigments, variable fluorescence (F_v/F_m) and chromophoric dissolved organic matter (CDOM) in CDOM-rich waters. The three-laser instrument configuration (375, 405, and 510 nm excitation) provides additional Fv/Fm measurements with 405 nm excitation, CDOM assessments in a broad concentration range, and potential for spectral discrimination between oil and CDOM fluorescence. The new measurement protocols, analytical algorithms and examples of laboratory and field measurements are discussed

Photo-physiological variability in phytoplankton chlorophyll fluorescence and assessment of chlorophyll concentration

Photo-physiological variability of in vivo chlorophyll fluorescence (CF) per unit of chlorophyll concentration (CC) is analyzed using a biophysical model to improve the accuracy of CC assessments. Field measurements of CF and photosystem II (PSII) photochemical yield (PY) with the Advanced Laser Fluorometer (ALF) in the Delaware and Chesapeake Bays are analyzed vs. high-performance liquid chromatography (HPLC) CC retrievals. It is shown that isolation from ambient light, PSII saturating excitation, optimized phytoplankton exposure to excitation, and phytoplankton dark adaptation may provide accurate in vivo CC fluorescence measurements (R2 = 0.90-0.95 vs. HPLC retrievals). For in situ or flow-through measurements that do not allow for dark adaptation, concurrent PY measurements can be used to adjust for CF non-photochemical quenching (NPQ) and improve the accuracy of CC fluorescence assessments. Field evaluation has shown the NPQ-invariance of CF/PY and CF(PY-1-1) parameters and their high correlation with HPLC CC retrievals (R2 = 0.74-0.96), while the NPQ-affected CF measurements correlated poorly with CC (R2 = -0.22)

FIRe glider: Mapping in situ chlorophyll variable fluorescence with autonomous underwater gliders

Nutrient and light availability regulate phytoplankton physiology and photosynthesis in the ocean. These physiological processes are difficult to sample in time and space over physiologically and ecologically relevant scales using traditional shipboard techniques. Gliders are changing the nature of data collection, by allowing a sustained presence at sea over regional scales, collecting data at resolution not possible using traditional techniques. The integration of a fluorescence induction and relaxation (FIRe) sensor in a Slocum glider allows autonomous high‐resolution and vertically‐resolved measurements of photosynthetic physiological variables together with oceanographic data. In situ measurements of variable fluorescence under ambient light allows a better understanding of the physical controls of primary production (PP). We demonstrate this capability in a laboratory setting and with several glider deployments in the Southern Ocean. Development of these approaches will allow for the in situ evaluation of phytoplankton light stress and photoacclimation mechanisms, as well as the role of vertical mixing in phytoplankton dynamics and the underlying physiology, especially in remote locations and for prolonged duration

Laser fluorescence analysis of phytoplankton across a frontal zone in the California Current ecosystem

Spatial variability of chlorophyll, phycobiliproteins, chromophoric dissolved organic matter and variable fluorescence (Fv/Fm) was analyzed across a deep-water density front in the Southern California Current Ecosystem using an Advanced Laser Fluorometer (ALF) calibrated to assess chlorophyll concentration (Cchl), total autotrophic carbon (AC) and Synechococcus carbon biomass (SYN). Three distinct autotrophic assemblages were identified. Fluorescence was found to be three to four times higher in cooler mesotrophic waters north of the front than in warm oligotrophic waters to the south. Northern waters were distinguished by a shallow pigment maximum dominated by a blue-water type of Synechococcus and by the presence of green-water Synechococcus and cryptophytes; only blue-water Synechococcus were detected at lower concentration south of the front. The highest Cchl and AC values, accompanied by elevated Fv/Fm and chlorophyll fluorescence per unit of Cchl, and minimal Synechococcus abundance, were found directly at the front in a 20–40 m deep layer dominated by diatoms. The covariation of Fv/Fm with nitrate concentration in this layer, along with the structural changes in the phytoplankton community, suggest that it had been generated by in situ processes rather than advection. Strong structural responses to the local hydrography were also revealed by high-frequency underway ALF surface sampling, which detected an abrupt transition from low to high SYN on the northern side of a sharp salinity gradient at the front. Synechococcus-specific phycoerythrin fluorescence (FPE12) and SYN were highly correlated in surface waters (R2= 0.95), while FPE12:SYN gradually increased with depth. Strong relationships were found for chlorophyll fluorescence versus Cchl (R2= 0.95) and AC (R2= 0.79)

Elevated surface chlorophyll associated with natural oil seeps in the Gulf of Mexico

Natural hydrocarbon seeps occur on the sea floor along continental margins, and account for up to 47% of the oil released into the oceans. Hydrocarbon seeps are known to support local benthic productivity, but little is known about their impact on photosynthetic organisms in the overlying water column. Here we present observations with high temporal and spatial resolution of chlorophyll concentrations in the northern Gulf of Mexico using in situ and shipboard flow-through fluorescence measurements from May to July 2012, as well as an analysis of ocean-colour satellite images from 1997 to 2007. All three methods reveal elevated chlorophyll concentrations in waters influenced by natural hydrocarbon seeps. Temperature and nutrient profiles above seep sites suggest that nutrient-rich water upwells from depth, which may facilitate phytoplankton growth and thus support the higher chlorophyll concentrations observed. Because upwelling occurs at natural seep locations around the world, we conclude that offshore hydrocarbon seeps, and perhaps other types of deep ocean vents and seeps at depths exceeding 1,000 m, may influence biogeochemistry and productivity of the overlying water column

Autonomous Ocean Measurements in the California Current Ecosystem

Event-scale phenomena, of limited temporal duration or restricted spatial extent, often play a disproportionately large role in ecological processes occurring in the ocean water column. Nutrient and gas fluxes, upwelling and downwelling, transport of biogeochemically important elements, predator-prey interactions, and other processes may be markedly influenced by such events, which are inadequately resolved from infrequent ship surveys. The advent of autonomous instrumentation, including underwater gliders, profiling floats, surface drifters, enhanced moorings, coastal high-frequency radars, and satellite remote sensing, now provides the capability to resolve such phenomena and assess their role in structuring pelagic ecosystems. These methods are especially valuable when integrated together, and with shipboard calibration measurements and experimental programs

Influence of the Amazon River Discharge on the Biogeography of Phytoplankton Communities in the Western Tropical North Atlantic

An Advanced Laser Fluorometer (ALF) capable of discriminating several phytoplankton pigment types was utilized in conjunction with microscopic data to map the distribution of phytoplankton communities in the Amazon River plume in May–June-2010, when discharge from the river was at its peak. Cluster analysis and Non-metric Multi-Dimensional Scaling (NMDS) helped distinguish three distinct biological communities that separated largely on the basis of salinity gradients across the plume. These three communities included an “estuarine type” comprised of a high biomass mixed population of diatoms, cryptophytes and green-water Synechococcus spp. located upstream of the plume, a “mesohaline type” made up largely of communities of Diatom-Diazotroph Associations (DDAs) and located in the northwestern region of the plume and an “oceanic type” in the oligotrophic waters outside of the plume made up of Trichodesmium and Synechococcus spp. Although salinity appeared to have a substantial influence on the distribution of different phytoplankton groups, ALF and microscopic measurements examined in the context of the hydro-chemical environment of the river plume, helped establish that the phytoplankton community structure and distribution were strongly controlled by inorganic nitrate plus nitrite (NO3 + NO2) availability whose concentrations were low throughout the plume. Towards the southern, low-salinity region of the plume, NO3 + NO2 supplied by the onshore flow of subsurface (∼80 m depth) water, ensured the continuous sustenance of the mixed phytoplankton bloom. The large drawdown of SiO3 and PO4 associated with this “estuarine type” mixed bloom at a magnitude comparable to that observed for DDAs in the mesohaline waters, leads us to contend that, diatoms, cryptophytes and Synechococcus spp., fueled by the offshore influx of nutrients also play an important role in the cycling of nutrients in the Amazon River plume