Fluorescence properties of natural phytoplankton populations

The cellular fluorescence of chlorophyll a in natural phytoplankton was measured during vertical profiling in marine coastal waters. The ratio of in situ fluorescence to chlorophyll a concentration, which was considered as an index of cellular fluorescence, varied over a wide range, with large changes occurring both within the water column and between profiling sites. The variations were caused in part by an inhibition in the fluorescence of cells exposed to intense sunlight. The inhibition, which occurred at irradiances exceeding 0.15 langley (ly)/min, led to diel fluctuations in the fluorescence of those phytoplankton near the sea surface. The remaining variations were independent of changes in temperature, but were unexplained. Both light-dependent and light-independent variations in cellular fluorescence will affect the accuracy of the continuous, fluorometric measurement of in vivo chlorophyll.     

Buoyancy of natural populations of marine phytoplankton

Buoyancy of natural populations of marine phytoplankton was studied in a fjord in western Norway during the diatom bloom and in autumn. The study was carried out under approximate in situ conditions by means of an apparatus described in the paper. During the spring bloom, positive buoyancy was observed only once. Sinking rates of individual fractions ranged from 0 to more than 9 m day^-1, and the mean sinking rates of the total chlorophyll content from 0 to at least 2. 2 m day^-1. The highest rates occurred in the post-bloom period, while sinking appeared negligible from the onset of the bloom up to its culmination. In autumn, the population was dominated by small, flagellated cells. Positive buoyancy, or upward migration, was then observed in two out of three experiments.     

Short-term variability of photosynthesis in natural marine phytoplankton populations

Harmonic regression analysis has been used to determine the short-term variability in the photosynthetic rate (mgC/mg chlorophyll a/h) of phytoplankton in three inlets of Japan. In natural water without large zooplankton present, the photosynthetic rate [log P=log (100xmgC/mg chlorophyll a/h)] can be expressed as (B+A cos T). Factor B represents the average photosynthetic rate, of which the maximum is usually designated as P _max, and Factor A corresponds to the slope of the regression line. The phase of the periodicity, represented by T, is adjusted to give the highest correlation: usually T is expressed as [360/24 x (local time + 4)] in degrees. The correlation between Factors A and B is very high (r=0.95, P<0.001), indicating that Factor A may depend upon Factor B (potential activity of chlorophyll a). Both Factors A and B decrease with decreasing irradiance, but the slope of each regression between Factor A and irradiance varies with season. Continuous darkness reduces the phase of the periodicity to one cycle a day when phytoplankton has multiple cycles of photosynthetic rate per day. Adequate nutrient supply from zooplankton regeneration may cause an increase in Factor B; however, excess density of zooplankton decreases Factor A.     

Effects of diurnal variations in phytoplankton photosynthesis obtained from natural fluorescence

Effects of diurnal variation in phytoplankton photosynthesis on estimating daily primary production (DPP) were examined using field data from Sagami Bay, Japan. DPP at 5 m depth was calculated from the continuous data of chlorophyll a (Chl a) and light intensity monitored by a natural fluorescence sensor with and without considering time-dependent changes in the photosynthesis–irradiance (P–E) relationship. Chl a could be estimated from natural fluorescence examining the variations in the quantum yield of fluorescence (φ _f) and Chl a-specific light absorption coefficient (a*_ph), and relating them to Chl a. The P–E relationship was determined by water sampling three times daily. A distinct diurnal pattern was observed for the maximum photosynthetic rate (P*_max), being maximal at noon, while periodicity of the maximum light utilization coefficient (α*) was less obvious. The actual DPP was calculated by interpolating the P–E parameters from those obtained at dawn, noon, and dusk. For comparison, DPP was calculated by fixing the P–E parameters as the constants measured at dawn, noon or dusk for a day. The difference from the actual DPP was small when the P–E parameters measured at dawn (3% on average) and noon (5%) were used as the constants for a day. The difference was largest when the values at dusk were used (−43%). The medium values of P*_max at dawn, its low values at dusk, and the fact that a major part of the DPP was produced around noon were responsible for these results. The present study demonstrates that measurement of the P–E parameters at dawn or noon can give a good estimation of DPP from natural fluorescence.     

Products of photosynthesis in natural populations of marine phytoplankton from the Gulf of Maine

We have measured the photosynthetic assimilation of ¹⁴C-carbon dioxide into (1) ethanol-soluble, (2) hot-trichloroacetic acid (TCA)-soluble (polysaccharide), and (3) protein fractions of natural populations of marine phytoplankton. Diurnal studies showed a continuing incorporation of carbon-14 into the protein fraction during hours of darkness. This was accompanied by a concomitant decrease in the proportion assimilated into polysaccharide. When incorporation was measured under constant experimental conditions, the pattern of photosynthesis did not vary from one time of day to another. At one station approximately 12 km south of Boothbay Harbor, the proportion of carbon entering protein showed marked seasonal changes. During the winter, approximately 10 to 20% of the fixed carbon was incorporated into protein. During the summer the value increased to 22 to 35%. Between these times, a transient high value of 37 to 47% of the fixed carbon entering protein coincided with the spring bloom. The increases in proportion incorporated into protein were largely paralleled by equivalent decreases in the polysaccharide fraction. The proportion of carbon incorporated into protein during photosynthesis also increased markedly at reduced light intensities. This increase occurred both when populations were incubated in neutral-density filters and when incubated at increasing depths in the photic zone. There was little consistent and significant difference between the neutral-density filters and depth in the water column, suggesting a minimal role for light quality. The extent of the increased relative rate of protein synthesis at the lower light intensities depended on the nutritional state of the phytoplankton. For example, summer populations from water containing low concentrations of inorganic nutrients responded less dramatically to reduced light intensities than did populations from nutrient-rich waters.     

The nature of the photosynthate in natural phytoplankton populations in relation to light quality

The relative concentrations of a variety of organic compounds in phytoplankton from different depths of the photic zone of Saanich Inlet and Indian Arm, British Columbia, Canada, were determined. In surface phytoplankton the greatest proportion of ¹⁴C in newly-formed organic compounds was in the ethanol-soluble fraction irrespective of the light intensity. The proportion of the label in the insoluble fraction increased with depth. Within the ethanol-soluble fraction the relative importance of carbohydrates decreased and amino acids increased with depth. Experiments in Indian Arm indicated that these observed changes were in response to light quality rather than intensity. The percentage release of dissolved organic carbon as a percentage of total fixation decreased with depth and was directly related to the size of the ethanol-soluble fraction.     

Significance of cellular nitrate content in natural populations of marine phytoplankton growing in shipboard cultures

Phytoplankton intracellular nitrate concentrations have been monitored in a 56-h experiment on a shipboard culture of surface sea water from an upwelling region. These measurements were related to parameters of biomass (particulate nitrogen) and nitrate assimilation using the ¹⁵N isotope technique and the nitrate reducase (NR) assay. The procedure for measuring cellular nitrate concentrations is described. This parameter exhibited diurnal variations, ranging from 3.1 to 20.6 ng-at nitrate per g-at particulate nitrogen, and could be correlated positively with NR activity. Nitrogen budgets show that NR activity represents only 12% of nitrate incorporation in organic phytoplankton material when nitrate is available in the sea water. However, upon depletion of the environmental nitrate (zero uptake), NR activity can fully account for the decrease of internal nitrate. From the results, it seems that internal nitrate content is a better index of nitrate consumption by marine phytoplankton than the external concentration of nitrate-nitrogen.     

Sinking-rate response of natural assemblages of temperate and subtropical phytoplankton to nutrient depletion

Phytoplankton assemblages were collected during spring blooms in 1982 in Washington State and in Hawaii. Sinking rate responses of these assemblages were examined under nitrate, phosphate, and silicate depletion. Ambient nutrient concentrations, chlorophyll concentrations, photosynthetic rates, sinking rates, and floristic compositions were determined. Under nutrient-replete conditions, the temperate assemblage, composed primarily of large centric diatoms, had a sinking rate of 0.96 m d^-1; sinking rates did not change appreciably over 4 d without nitrate. Without phosphate or silicate, the sinking rates remained constant for 3 d and then increased after biomass indices began to decline. These findings illustrate the potential importance of phosphate or silicate depletion to the sedimentation of spring-bloom diatom populations. The subtropical assemblage, composed primarily of diatoms, coccolithophorids, and dinoflagellates, had an initial sinking rate of 0.22 m d^-1 and did not display substantial sinking rate changes in the absence of nitrate, phosphate or silicate. Floristic data consistently showed a proliferation of pennate diatoms, which had lower settling rates than centric diatoms. Growth and sedimentation patterns indicated a competitive advantage for pennate diatom components of subtropical assemblages; this in turn may limit phytoplankton sedimentation losses in such ecosystems.     

Persistent diel rhythms in the chlorophyll fluorescence of marine phytoplankton species

The diel periodicities of in-vivo chlorophyll fluorescence and DCMU-enhanced chlorophyll fluorescence of 47 marine phytoplankton species were examined for 2 d in a 14 h L:10 h D light: dark cycle and then in continous light for another 2 to 3 d. Almost all phytoplankton species exhibit a more rapid increase in in-vivo fluorescence and DCMU-enhanced fluorescence during the light phase than during the dark phase. About one-half of the species examined exhibited persistent diel rhythms in continous light, indicating the operation of a biological clock. No phylogenetic or habitat related trends as to which species exhibited persistent rhythms were apparent. Of the phyla of eukaryotic phytoplankton adequately examined, none lacked biological clocks. Contrary to past hypotheses, some phytoplankton species maintain a persistent diel rhythm in a constant environment while reproducing at a rate greater than one division per day.     

Photosynthesis and chlorophyll a fluorescence rhythms of marine phytoplankton

Circadian rhythms in photosynthesis were defined in field populations of phytoplankton. Measurements of carbon-dioxide fixation rates demonstrated that a diurnal periodicity of photosynthesis in samples incubated under natural light-dark (LD) cycles also were observed to continue in similar samples which had been photoadapted to constant dim light (LL) for 48 h. These changes in photosynthetic rates preceded sunset and sunrise, had daily amplitudes that ranged from 1.5 to 2.0, appeared to be independent of light-intensity, and displayed maxima about midday, while rates of dark fixation of carbon dioxide and the photosynthetic pigment content per cell were constant over the circadian cycle. Similar rhythmicity also was detected in room-temperature (22°C) chlorophyll a fluorescence yield, in both the obsence and presence of the photosynthesis inhibitor DCMU [3-(3,4-dichlorophenyl)-1, 1-dimethylurea]. However, the magnitude and timing of the fluorescence rhythm maxima seem to depend on wavelengths monitored and, in part, on the measuring technique used. Also, the circadian changes in the fluorescence intensity were abolished at low temperature (-60°C), and the shape of the emission spectra of chlorophyll fluorescence of cells in LD and LL did not change over time. The significance of the fluorescence rhythms with regard to chlorophyll a determinations and photosynthetic rates is discussed. It was concluded that there was sufficient similarity between circadian rhythms of photosynthesis in natural phytoplankton populations and in laboratory cultures of dinoflagellates to suggest that the mechanism of regulation may be the same for both of them.     

Spatial heterogeneity of phytoplankton populations in estuarine surface microlayers

Phytoplankton populations are concentrated in surface microlayer waters of the York River and Mobjack Bay, Virginia, USA. The degree of vertical heterogeneity was variable and seemed most dependent on weather conditions (vertical mixing) and total phytoplankton cell densities. Vertical stratification also exhibited some taxocoenotic dependency. Structurally, the phytoplankton communities of the surface microlayer were less diverse than subsurface communities. In general, values of informational diversity (H'), evenness (J), and richness (S and r) are lowest in the surface microlayer and increase with depth (to 3.0 m). Mechanisms which structure and maintain vertical heterogeneity are discussed.This work was supported in part by funds from the National Science Foundation Research Applied to National Needs Program grants to the Virginia Institute of Marine Science and the Chesapeake Research Consortium.Contribution No. 791 of the Virginia Institute of Marine Science, and Contribution No. 62 of the South Carolina Marine Resources Center.     

浮游植物叶绿素a测定方法比较

选取规范方法、超声波法、反复冻融法、延时提取法、热丙酮法、丙酮加热法、热乙醇法、混合溶剂法8种常见的浮游植物叶绿素a的测定方法,以实验室培养的微囊藻(Microcystis aeruginosa)水华样品为研究对象进行叶绿素a含量的测定.从叶绿素a提取的完全性、测定数据的稳定性、试验操作的简便性等方面对8种方法进行评价,并对叶绿素a结果测量的单色法和三色法进行分析讨论.试验结果表明,选取的8种测定方法中,包括国家标准方法在内的大多数方法,存在操作耗时长、过程复杂、叶绿素提取不完全的缺点;而丙酮加热法测量叶绿素a的提取效率高、数据稳定性好,操作耗时短、简便,可推荐作为一种常用的浮游植物叶绿素a的测量方法;三色法和单色法测定方法的选取应综合考虑.     

DCMU-enhanced fluorescence as an index of photosynthetic activity in phytoplankton

Tests have been carried out, both on phytoplankton cultures and in the field, on the relation between the ratio of 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU)-enhanced to normal fluorescence (F _D /F _N ) and the specific photosynthetic rate (P/B). In all cases observed, significant linear correlations were found between these two values. Differences in this relation were observed according to species and physiological conditions. Nutrient stress that occurs in batch cultures decreases both F _D /F _N and P/B, while lowered light intensity has a different effect on both, increasing F _D /F _N and decreasing P/B. This is interpreted as indicating that F _D /F _N is an index of photosynthetic efficiency at different light levels and an indicator of the specific photosynthetic rate (P/B) when physiological conditions vary at a given light intensity. The practical use of DCMU-enhanced fluorometry to estimate instantaneous P/B values in the field is discussed, stressing the frequent calibrations needed, as in all in situ fluorometry studies.     

Mesocosm studies of DCMU-enhanced fluorescence as a measure of phytoplankton photosynthesis

When measurements of in-vivo fluorescence are used to estimate photosynthesis in the field, the marked temporal and spatial variations in phytoplankton populations, and their nutrient and light histories, have produced varied results. Natural phytoplankton populations in large, flow-through mesocosms with different controlled nutrient and sewage sludge additions were sampled weekly from June to September 1984. Good correlations were observed between the increase in fluorescence upon the addition of DCMU (F) and both in-situ production and the parameters (P_m and ) of the photosynthesis-irradiance curve for these phytoplankton populations. Good correlations were also obtained between DCMU-enhanced fluorescence (F_DCMU) and chlorophyll a concentrations. The relationship between F and in-situ ¹⁴C production was consistent among mesocosms even in the face of major shifts from diatom-dominated to dinoflagellate-dominated populations. On the other hand, the F_DCMU:Chl a relationship was significantly different between mesocosms and related to species composition. It was concluded that F offers the possibility of rapidly and accurately indexing both in-situ production and the photosynthetic capacity of mixed phytoplankton populations.This study was supported by EPA cooperative agreement 810265-03 and NOAA grant NA-83-ABD-00008     

Assessment of simultaneous uptake of nitrogenous nutrients (15N) and inorganic carbon (13C) by natural phytoplankton populations

The simultaneous uptake of nitrogenous nutrients and inorganic carbon was measured in shipboard incubations of natural phytoplankton populations, using tracer additions of ¹³C-bicarbonate and ¹⁵N-labelled nitrogenous substrates. From March 1991 through March 1992, three stations on the Scotian Shelf (eastern Canada) were sampled monthly at ten depths in the euphotic zone. Additions of labelled nitrogen compounds ranged between 0.5 and 98% of ambient concentrations. Most of the C/N (at/at) uptake ratios were lower than the Redfield ratio, suggesting that nitrogen was not limiting. The fixation of carbon with and without addition of nitrate, ammonium or urea was generally similar. Some samples presented significant differences in carbon uptake rate between the four treatments, but these differences were not related to nitrogen enrichment (percent or nitrogen species). Given these results, the double-labelling method appears to be a reliable tool for measuring the simultaneous uptake of carbon and nitrogen by natural phytoplankton.     

Diel photosynthetic production of cellular organic matter in natural phytoplankton populations,measured with13C and gas chromatography/mass spectrometry

Diel photosynthetic production of water-extractable and residual carbohydrates in natural phytoplankton populations was investigated by analyzing monosaccharide components with combined gas chromatography/mass spectrometry (GC/MS) and¹³C tracer methods. The applicability of this technique for measuring the photosynthetic production of natural phytoplankton populations was examined. Phytoplankton was collected in August 1984 from Kinu-ura Bay, Japan, and incubated under natural light and dark conditions for 24 h. Cell density and concentration of chlorophylla increased about four- and three-fold, respectively, during the experimental period. Production of glucose, galactose, mannose, rhamnose, ribose, fucose, xylose and arabinose was measured in the water-extractable and residual carbohydrate fractions of the phytoplankton. Most of the monosaccharides in both fractions were produced the monosaccharides in both fractions were produced mainly during the daytime. Glucose was a major component in the water-extractable carbohydrate fraction produced by the phytoplankton, and decreased markedly at night. Thus, most of the glucose in the water-extractable carbohydrates seemed to be a constituent of the storage glucan of algal cells. Most monosaccharide components of the water-extractable carbohydrate fraction except glucose did not decrease significantly at night. Ribose of the water-extractable carbohydrate fraction was synthesized at a maximum rate in the morning, prior to the other monosaccharide constituents of this fraction. The major monosaccharide constituent in the residual carbohydrate fraction produced by the phytoplankton was glucose, which decreased significantly at night. Diel change in the production of monosaccharide constituents in residual carbohydrates indicated wide variability in the monosaccharide composition of cell-wall material in algae. The ratios of production of water-extractable and residual carbohydrates to POC production ranged from 8.8 to 28% and from 3.7 to 5.9%, respectively, throughout the daytime and nighttime.     

In situ characterization of phytoplankton from vertical profiles of fluorescence emission spectra

Vertical profiling of the upper ocean with a laser/fiber optic fluorometer enabled the determination of fluorescence emission spectra of photosynthetic pigments over small vertical scales. Simultaneous acquisition of phycoerythrin (PE) and chlorophyll (chl) emission spectra allowed in situ differentiation between PE-containing cells (cryptomonads and cyanobacteria) and other chl-containing autotrophs. Further, fluorescence spectral peak shifts associated with different species of PE-containing cells resulted in even finer scale in situ taxonomic differentiation. We found that the phycoerythrin fluorescence emission maxima shifted from 578 nm near the surface, to 585 m at the base of the shallow thermocline (30% light level), and to 590 nm below the thermocline at the base of the euphotic zone (1% light level). These shifts in peak emission coincided with a taxonomic change in the PE-containing cells (as determined from analysis of discrete bottle samples) from a greater proportion of Synechococcus spp. in the upper water column to a greater proportion of cryptomonads at the base of the euphotic zone. These results indicate that the composition of the phytoplankton assemblage may be assessed in situ without sample collection.     

Experiments on resuspension of natural microphytobenthos populations

Laboratory experiments on resuspension of benthic diatoms were performed in 1988 on intact cores of sediment obtained from two transects in the Ems estuary (Dutch Wadden Sea, NW Europe). Diatoms were most abundant in sediment from sheltered stations. In the experiments, which were performed in a small carousel system, the percentage of resuspended diatoms increased concomitantly with angular velocity of the agitating paddles. Maximum microphytobenthos resuspension in terms of cell numbers was up to 45% of the total population present in the 0.5-cm top layer, and up to 11% in terms of chlorophylla. Maximum sediment resuspension was 6%. The results show that the presence of a diatom film (numerous cells on the sediment surface) increases sediment stability, thereby suppressing resuspension of sediment and diatoms. However, if the sediment contains many fine particles and much detritus, sediment stability decreases, leading to increased resuspension.These results support the contention that low detritus content and large numbers stabilize the surface layer of tidal flats, whereas high detritus content and fewer cells (<2 × 10⁵ cm^–2) do not. The stabilization is thought to be dependent on a combination of differences in sediment consolidation and in bottom roughness caused by the presence or absence of diatom films.     

Vertical distribution of phytoplankton during investigations of a natural surface film

An interdisciplinary research program (Kombiniertes Oberflächenfilmprojekt KOFF), in the North Sea area about 75 km west of the Island of Sylt, is directed towards gaining further knowledge on the biological importance of the natural surface film. The investigations include the water body below the film as well as dynamic processes at the surface. Persistence of calm weather conditions (cloudless sky) before and during the investigations permitted observations of the diurnal species-specific vertical migration of Dinophyceae. Two groups could be distinguished: (1) Prorocentrum micans and Ceratium furca; (2) C. horridum, C. macroceros and C. fusus. The active accumulation of P. micans in the slick was verified by a significant correlation coefficient—linearly correlated with the time of day. The results presented reveal continuity between surface film and underlying water column.This work was supported by the Sonderforschungsbereich (SFB) 94, Hamburg, of the Deutsche For-schungsgemeinschaft.