The soluble fluorescence in the open sea: Distribution and ecological significance in the equatorial Atlantic Ocean

The red fluorescence of filtered sea water has been measured on 216 samples in the 0–150 m layer of the equatorial Atlantic Ocean.Soluble fluorescence is maximum where chlorophyll a and in vivo fluorescence are maximum, but the percentage of soluble fluorescence, (soluble fluorescence/in vivo fluorescence) × 100, is minimum at these levels; in recently upwelled waters of the equatorial divergence, the percentage of soluble fluorescence is equal to 10 in the 0–20 m layer and regularly increases to 60 or more at 100–150 m; in the nitrate depleted mixed layer of a convergence it averages 30, decreases to 15 in the thermocline maximum of chlorophyll a, and again reaches 60 in deep waters.A significant positive correlation has been found between the percentage of soluble fluorescence and the amount of phaeophytin, and soluble fluorescence in the open sea is thought to be the result of the degradation and release of chloroplastic products by aged or grazed phytoplankton populations. Low values (< 20) of the percentage soluble fluorescence indicate the presence of healthy phytoplankton cells, whereas high values (> 30) are evidence of unfavourable growth conditions (e.g., limiting nutrients or darkness) or high grazing pressure.The simultaneous measurement of in vivo fluorescence and soluble fluorescence is a method of obtaining valuable information rapidly on the physiological state of the phytoplankton population in the water column.     

Tidal and diel fluctuations in the temporal concentrations of chlorophyll a and pheophytin at a station monitoring a high-marsh creek

The temporal distribution of chlorophyll a and pheophytin at a transect monitoring the flow at a high-marsh creek was investigated. The observed fluctuations in chlorophyll a concentration consisted of complex, superimposed, tidal and diel rhythms; pheophytin variability, on the other hand, was controlled by the tides. Transport measurements and correlation analyses supported the hypothesis that tidal forces have a major influence on the temporal fluctuation of chlorophyll a and phaeophytin concentrations in high-marsh creeks. The data indicate that it is important to consider tidal flux when designing programs to study seasonal effects, primary productivity, and phytoplankton species composition.     

The use of acetone and methanol in the estimation of chlorophyll in the presence of phaeophytin

(1)Where grinding or maceration of plant tissue is impractical, immersion in methanol should be used. (2)The adaption to aqueous methanol of existing techniqus for distinguishing chlorophyll and phaeophytin in aqueous acetone was studied in detail. In methanol the spectra of both phaeophytin a and b were found to be pH sensitive. A method developed involving changes in absorbance at 665 nm is less precise than similar methods using acetone. (3)The spectra of phaeophytin b in methanol at different pH levels are anomalous. Changes in absorbance between 440 and 410 nm cannot be used for the estimation of phaeophytin. (4)Plant pigments can be transferred from methanol to aqueous acetone without degradation of chlorophyll. Modified standard techniques may then be used to measure chlorophyll a and phaeophytin a content.     

Enzymatic degradation of chlorophyll a by marine phytoplankton in vitro

The enzymatic degradation of chlorophyll a and the formation of chlorophyllide a, phaeophytin a, and phaeophorbide a were detected in vitro in several species of marine phytoplankton. Loss of phytol and Mg²⁺ were found to be catalysed by chlorophyllase and a magnesium-releasing enzyme, respectively. The activities of the two enzymes could be distinguished from each other by inhibiting with Mg²⁺ and/or p-chloromercurobenzoate. Both enzymes are activated by cell disintegration. Degradation products were not detected spectrophotometrically in vivo. Additionally, in some species, chlorophyll a was degraded to products which do not absorb visible light.     

DIFFERENCES BETWEEN IN VIVO ABSORPTION AND FLUORESCENCE EXCITATION SPECTRA IN NATURAL SAMPLES OF PHYTOPLANKTON

The fluorescence excitation spectrum of live phytoplankton cells represents the portion of light absorbed that has been effectively transferred to chlorophyll a of photosystem II, whereas light absorbed by photoprotective pigments will not lead to fluorescence. Therefore, the in vivo fluorescence excitation spectrum of phytoplankton has been used as a proxy for the action spectrum of phytoplankton in computations of primary production in the ocean. The distribution of chlorophyll a between photosystems, as well as variations in the pathway of energy inside the photosynthetic membrane, can also influence the fluorescence excitation spectrum. In this study, we investigated the contribution of photoprotective pigments to the differences found between in vivo absorption and fluorescence excitation spectra of phytoplankton measured during two cruises: one from Las Islas Canarias to Nova Scotia and another in the Labrador Sea. A comparison of normalized fluorescence excitation and absorption spectra showed high variability in the difference between absorption and fluorescence in the blue region of the spectrum for samples from the two cruises. This difference was not entirely correlated with the concentration of photoprotective carotenoids. In this paper, results are interpreted in terms of differences in pigment composition and known patterns of energy distribution in the photosystems of different algal groups.     

In situ and remote-sensed chlorophyll fluorescence as indicator of the physiological state of phytoplankton near the Isles Kerguelen (Southern Ocean)

Shipboard and remote-sensed Chlorophyll fluorescence were determined in the natural phytoplankton assemblage above the iron-enriched Kerguelen Plateau and the adjacent high-nutrient, low-Chlorophyll open Southern Ocean. The variance between fluorescence yield and photosynthetic efficiency was determined in combination with Chlorophyll a concentrations, irradiance and phytoplankton species distribution. A co-variance between the fluorescence measurements would allow the refinement of remote-sensing primary production algorithms. Distinct differences were found in photosynthetic efficiency and water-leaving fluorescence, with relatively high values for the Kerguelen Plateau and low values in the open ocean, reflecting the differences in Chlorophyll a concentrations. The co-variance of the fluorescence properties suggested that remote-sensed fluorescence measurements could be used to infer differences in the physiological state of the phytoplankton, hence primary production. Fluorescence yield, however, did not show the differences in the research area, most likely due to the low signal and the diurnal variation in water-leaving fluorescence.     

Microflow Cytometer for optical analysis of phytoplankton

Analysis of the intrinsic fluorescence profiles of individual marine algae can be used in general classification of organisms based on cell size and fluorescence properties. We describe the design and fabrication of a Microflow Cytometer on a chip for characterization of phytoplankton. The Microflow Cytometer measured distinct side scatter and fluorescence properties of Synechococcus sp., Nitzschia d., and Thalassiosira p.; measurements were confirmed using the benchtop Accuri C6 flow cytometer. The Microflow Cytometer proved sensitive enough to detect and characterize picoplankton with diameter approximately 1 μm and larger phytoplankton of up to 80 μm in length. The wide range in size discrimination coupled with detection of intrinsic fluorescent pigments suggests that this Microflow Cytometer will be able to distinguish different populations of phytoplankton on unmanned underwater vehicles.     

Flow cytometry: instrumentation and application in phytoplankton research

In flow cytometry, light scattering and fluorescence of individual particles in suspension is measured at high speed. When applied to planktonic particles, the light scattering and (auto-)fluorescence properties of algal cells can be used for cell identification and counting. Analysis of the wide size spectrum of phytoplankton species, generally present in eutrophic inland and coastal waters, requires flow cytometers specially designed for this purpose. This paper compares the performance in phytoplankton research of a commercial flow cytometer to a purpose built instrument. It reports on the identification of phytoplankton and indicates an area where flow cytometry may supersede more conventional techniques: the analysis of morphological and physiological characteristics of subpopulations in phytoplankton samples.     

Surplus photosynthetic antennae complexes underlie diagnostics of iron limitation in a cyanobacterium

Chlorophyll fluorescence from phytoplankton provides a tool to assess iron limitation in the oceans, but the physiological mechanism underlying the fluorescence response is not understood. We examined fluorescence properties of the model cyanobacterium Synechocystis PCC6803 and a ΔisiA knock-out mutant of the same species grown under three culture conditions which simulate nutrient conditions found in the open ocean: (1) nitrate and iron replete, (2) limiting-iron and high-nitrate, representative of natural high-nitrate, low-chlorophyll regions, and (3) iron and nitrogen co-limiting. We show that low variable fluorescence, a key diagnostic of iron limitation, results from synthesis of antennae complexes far in excess of what can be accommodated by the iron-restricted pool of photosynthetic reaction centers. Under iron and nitrogen co-limiting conditions, there are no excess antennae complexes and variable fluorescence is high. These results help to explain the well-established fluorescence characteristics of phytoplankton in high-nutrient, low-chlorophyll ocean regions, while also accounting for the lack of these properties in low-iron, low-nitrogen regions. Importantly, our results complete the link between unique molecular consequences of iron stress in phytoplankton and global detection of iron stress in natural populations from space.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> extraction from freshwater algae — a reevaluation

In this study, two extracting methods (sonication and dispersing) and three solvents (90% acetone, N,N′-dimethylformamide and methanol) were compared for their ability to extract chlorophyll a of freshwater phytoplankton. Measurements were performed with both spectrophotometry and high-performance liquid chromatography. Results showed that (i) cell disruption is essential and that (ii) the method of cell disruption and solvent applied differed significantly. Dispersing in acetone surpassed all other combinations. Sonication in N,N′-dimethylformamide was found less effective. N,N′-dimethylformamide and methanol seem to promote the formation of degradation products (chlorophyllide a, allomer, epimer and phaeophytin a) which lead to overestimates of chlorophyll a of about 10% by means of spectrophotometry.     

Estimation of Primary Productivity by Chlorophyll a in vivo Fluorescence in Freshwater Phytoplankton

Primary productivity in marine waters is widely estimated by the measurements of ¹⁴C incorporation, the underwater light climate, and the absorption spectra of phytoplankton. In bio-optical models the quantum efficiency of carbon fixation derived from ¹⁴C incorporation rates, the photosynthetically absorbed radiation derived from the underwater light climate, and the phytoplankton absorption spectra are used to calculate time- and depth-integrated primary productivity. Due to the increased sensitivity of commercially available fluorometers, chlorophyll a in vivo fluorescence became a new tool to assess the photosynthetic activity of phytoplankton. Since fluorescence data yield only relative photosynthetic electron transport rates, a direct conversion into absolute carbon fixation rates is not possible. Here, we report a procedure how this problem can be adressed in freshwater phytoplankton. We adapted a marine bio-optical model to the freshwater situation and tested if this model yields realistic results when applied to a hypertrophic freshwater reservoir. Comparison of primary productivity derived from ¹⁴C incorporation to primary productivity derived from Chl a fluorescence showed that the conversion of fluorescence data into carbon fixation rates is still an unsolved problem. Absolute electron transport rates calculated from fluorescence data tend to overestimate primary production. We propose that the observed differences are caused mainly by neglecting the package effect of pigments in phytoplankton cells and by non-carbon related electron flow (e.g., nitrogen fixation). On the other hand, the ¹⁴C incorporation rates can be artificially influenced by "bottle effects", especially near the water surface, where photoinhibition, photorespiration, and Mehler reaction can play a major role.     

A reduced model of the fluorescence from the cyanobacterial photosynthetic apparatus designed for the in situ detection of cyanobacteria

Fluorometric determination of the chlorophyll (Chl) content of cyanobacteria is impeded by the unique structure of their photosynthetic apparatus, i.e., the phycobilisomes (PBSs) in the light-harvesting antennae. The problems are caused by the variations in the ratio of the pigment PC to Chl a resulting from adaptation to varying environmental conditions. In order to include cyanobacteria in fluorometric analysis of algae, a simplified energy distribution model describing energy pathways in the cyanobacterial photosynthetic apparatus was conceptualized. Two sets of mathematical equations were derived from this model and tested. Fluorescence of cyanobacteria was measured with a new fluorometer at seven excitation wavelength ranges and at three detection channels (650, 685 and 720 nm) in vivo. By employing a new fit procedure, we were able to correct for variations in the cyanobacterial fluorescence excitation spectra and to account for other phytoplankton signals. The effect of energy-state transitions on the PC fluorescence emission of PBSs was documented. The additional use of the PC fluorescence signal in combination with our recently developed mathematical approach for phytoplankton analysis based on Chl fluorescence spectroscopy allows a more detailed study of cyanobacteria and other phytoplankton in vivo and in situ.     

PHOTOINHIBITION OF TEMPERATE LAKE PHYTOPLANKTON BY NEAR-SURFACE IRRADIANCE: EVIDENCE FROM VERTICAL PROFILES AND FIELD EXPERIMENTS1

To evaluate the in situ occurrence of phytoplankton photoinhibition, the light-mediated depression of chlorophyll in vivo fluorescence (IVF) and of the cellular fluorescence capacity (CFC) of phytoplankton was determined in three southeastern United States reservoirs. Vertical profiles of a fluorescence depression index (FDI) and of the CFC for reservoir phytoplankton showed that near-surface photoinhibition of fluorescence properties occurred in association with high surface irradiance and weak vertical mixing of the water column. To characterize the time scales of photochemical and photosynthetic responses to and recovery from exposure to supraoptimal light intensity, phytoplankton IVF responses and ¹⁴C-fixation rates were measured infield experiments. Phytoplankton chlorophyll IVF, CFC, and photosynthetic ¹⁴C fixation were rapidly (20–40 min) depressed when reservoir phytoplankton were exposed to surface irradiances (1700–2000 μE·m^?2·s^?1). Light-mediated increases in the FDI declined rapidly (20–40 min) to pre-exposure levels during a subsequent low-light (75–200 μE·m^?2·s^?1) period, but CFC and ¹⁴C fixation recovered more slowly (>40 min). Exposure of reservoir phytoplankton to a light-intensity gradient revealed both intensity and time thresholds for IVF and CFC depression. Phytoplankton photochemical responses to bright light operate on time scales that, in conjunction with vertical mixing, should limit the occurrence of photoinhibition to extreme irradiance environments. Our results support the hypothesis that the photoinhibition of phytoplankton productivity occurs less commonly than is indicated by fixed-depth incubation measurements.     

The influence of wind-induced mixing on the vertical distribution of buoyant and sinking phytoplankton species

In this study we exploit recent advances in high-resolution autonomous monitoring to investigate the impact of short-term variations in wind-induced mixing on the surface biomass and vertical distribution of buoyant and sinking phytoplankton species. An autonomous platform (the Automatic Water Quality Monitoring Station) moored in a Mediterranean reservoir provided minute-by-minute records of wind speed and the phytoplankton fluorescence during winter and summer. This information was then used here to quantify the impact of short-term changes in the weather on the vertical distribution of diatoms and cyanobacteria. Additionally, we apply an empirical model to determine the extent of entrainment of diatoms and cyanobacteria within the turbulent upper layers of the water column. During winter, the surface time series of fluorescence was positively correlated with the short-term variations in wind speed. In contrast, during the summer, fluorescence was negatively correlated with wind speed. In the latter case, turbulence overcame the flotation velocity of buoyant cyanobacteria, thus homogenizing their vertical distribution and decreasing surface biomass. In both cases, the dynamic response of surface phytoplankton biomass to short-term changes in wind stress was rapid, within the minute scale. As far as we know from the literature, this is the first study in which the interaction between wind stress and surface phytoplankton fluorescence has been quantified on such a fine temporal scale. Finally, relevance for forecasting and reservoir management is pointed out.     

Phytoplankton patchiness in Winam Gulf,Lake Victoria: a study using principal component analysis of in situ fluorescent excitation spectra

1. In order to characterise phytoplankton patchiness at fine scales, a profiling multiwavelength fluorometer was cast at numerous locations throughout Winam Gulf in Lake Victoria to measure fluorescent excitation spectra, which are indicators of both phytoplankton diversity and coloured dissolved organic matter (CDOM). 2. Processing the spectral data with principal component analysis (PCA) revealed that linear combinations of four fundamental ‘base’ spectra could explain almost all of the variation in spectral measurements. Three of the base spectra were associated with spatially distinct patches of phytoplankton containing different species assemblages, while the fourth base spectrum was due to CDOM fluorescence. 3. The locations of the phytoplankton patches were traced to the south‐east of Winam Gulf, the western end of the Rusinga Channel and the open waters of Lake Victoria adjacent to Winam Gulf, respectively. The high CDOM fluorescence was traced mainly to relatively deep water in the Rusinga Channel. 4. The phytoplankton and CDOM patchiness were interpreted in the context of physical and chemical gradients that were measured at the site at the same scale as the spectral data. Strong relationships were found between the gradients in spectral data and other environmental variables, which suggested several underlying explanations for the phytoplankton and CDOM patchiness.     

Phytoplankton dynamics in relation to hydrography, nutrients and zooplankton at the onset of sea ice formation in the eastern Weddell Sea (Antarctica)

The quantitative and qualitative distribution of phytoplankton was investigated along five North–South transects in the eastern Weddell Sea during the transition from late autumn to winter. Relationships with the regional hydrography, progressing sea ice coverage, nutrient distribution and zooplankton are discussed and compared with data from other seasons. To the north of the Antarctic Slope Front (ASF) a remnant temperature minimum layer was found above the primary pycnocline throughout summer. Surface waters had not entirely acquired typical winter characteristics. While temperature was already in the winter range, this was not the case for salinity. Highest biomass of phytoplankton, with the exception of the first transect, was found in the region adjoining the ASF to the north. Absolute chlorophyll a (Chl a) concentrations dropped from 0.35 to 0.19 g l^–1 . Nutrient pools exhibited a replenishing tendency. Ammonium concentrations were high (0.75–2 mol l^–1), indicating extensive heterotrophic activity. The phytoplankton in the ASF region was dominated by nanoflagellates, particularly Phaeocystis spp.. North of the ASF the abundance of diatoms increased, with Fragilariopsis spp., F. cylindrus and Thalassiosira spp. dominating. Community structure varied both due to hydrographical conditions and the advancing ice edge. The phytoplankton assemblage formed during late autumn were very similar to the ones found in early spring. A POC/PON ratio close to Redfield, decreasing POC concentration and a high phaeophytin/Chl a ratio, as well as a high abundance of mesozooplankton indicated that a strong grazing pressure was exerted on the phytoplankton community. A comparison between primary production (PP) in the water column and the sea ice showed a shift of the major portion of PP into the ice during the period of investigation.     

The use of spectral fluorescence methods to detect changes in the phytoplankton community

In vivo fluorescence methods are efficient toolsfor studying the seasonal and spatial dynamics ofphytoplankton. Traditionally the measurements are madeusing single excitation-emission wavelengthcombination. During a cruise in the Gulf of Riga(Baltic Sea) we supplemented this technique bymeasuring the spectral fluorescence signal (SFS) andfixed wavelength fluorescence intensities at theexcitation maxima of main accessory pigments. Thesemethods allowed the rapid collection of quantitativefluorescence data and chemotaxonomic diagnostics ofthe phytoplankton community. The chlorophylla-specific fluorescence intensities (R) and thespectral fluorescence fingerprints were analysedtogether with concentrations of chlorophyll a indifferent algal size-groups, phytoplankton biomass andtaxonomic position. The lower level of R in thesouthern gulf was related to the higher proportion ofcyanobacteria relative to total biomass and the lowerabundance of small algae. The phycoerythrinfluorescence signal was obviously due to the largecyanobacteria. The basin-wide shift in the shape ofchlorophyll a excitation spectra was caused bythe variable proportions of differently pigmentedcyanobacteria, diatoms and cryptomonads. This revised version was published online in July 2006 with corrections to the Cover Date.     

Seasonal variability of phytoplankton at Varna Bay (Black Sea)

Intrinsic fluorescence and SDS-PAGE analysis were employed to study the seasonal qualitative and quantitative changes of phytoplankton composition at Varna Bay (Black Sea). Variation in the maximum emission wavelength (lambda(max)) of the phytoplankton proteins (398 nm in the summer and 340 nm in the spring) was observed. In addition, a decrease in fluorescence intensity, and a shift in lambda(max) as a result of changes in phytoplankton protein stability, according to the season, was noted. Similarly, SDS-PAGE analysis showed different protein patterns for each season, for example in summertime the major protein constituents were of 14, 37, 48 and 70 kDa, while in the springtime the sizes ranged between 38 and 48 kDa. In general, higher carbohydrate and protein contents correlated with larger phytoplankton biomass found during the summer. The dominant species, the Bacillariophyceae and Dinophyceae, were found to be present in the water body in an alternate pattern. All of these changes could be accounted for by the adaptation of the organism to seasonal variations that modify the sea environment at Varna Bay.     

The influence of the accuracy of sampling and processing methods on the parameters of the phytoplankton community of Rybinsk reservoir

The influence of the sampling and processing-method accuracy on the bioindication parameters of the taxonomic and size structure of the phytoplankton community, fluorescence, and saprobic index has been studied in Rybinsk Reservoir. The studied parameters have been ranked in accordance to their sensitivity to the method accuracy. The comparative analysis of the relative errors of the studied parameters has been performed for Rybinsk Reservoir and the White Sea phytoplankton communities. Recommendations about the most useful parameters to assess the status of the phytoplankton community are given.     

Photoadaptation in marine phytoplankton : changes in spectral absorption and excitation of chlorophyll a fluorescence

The optical properties of marine phytoplankton were examined by measuring the absorption spectra and fluorescence excitation spectra of chlorophyll a for natural marine particles collected on glass fiber filters. Samples were collected at different depths from stations in temperate waters of the Southern California Bight and in polar waters of the Scotia and Ross Seas. At all stations, phytoplankton fluorescence excitation and absorption spectra changed systematically with depth and vertical stability of the water columns. In samples from deeper waters, both absorption and chlorophyll a fluorescence excitation spectra showed enhancement in the blue-to-green portion of the spectrum (470-560 nm) relative to that at 440 nm. Since similar changes in absorption and excitation were induced by incubating sea water samples at different light intensities, the changes in optical properties can be attributed to photoadaptation of the phytoplankton. The data indicate that in the natural populations studied, shade adaptation caused increases in the concentration of photosynthetic accessory pigments relative to chlorophyll a. These changes in cellular pigment composition were detectable within less than 1 day. Comparisons of absorption spectra with fluorescence excitation spectra indicate an apparent increase in the efficiency of sensitization of chlorophyll a fluorescence in the blue and green spectral regions for low light populations.