Methodology of light response curves: application of chlorophyll fluorescence to microphytobenthic biofilms

The light response curve methodology for microphytobenthic biofilms was studied by comparing the two most usual approaches used in pulse amplitude modulated (PAM) fluorometry. The non-sequential light curve (N-SLC) method is characterized by independent measures of the photosynthetic activity across a light gradient whereas the rapid light curve (RLC) method consists of successive measures on the same sample exposed to a stepwise increase of light intensities. Experiments were carried out on experimental microphytobenthic biofilms prepared from natural assemblages and acclimated to dark conditions. In preliminary experiments, N-SLCs were constructed from fluorescence induction curves performed at 12 different photon flux densities (PFDs). A minimum of 50 s of illumination was necessary to reach a stable light response curve; shorter illumination times resulted in underestimating the physiological parameters (α the light utilization coefficient in light-limited conditions and rETR_max the maximum rate of photosynthesis efficiency) of the light response curve. For the comparison between N-SLCs and RLCs, the same time of illumination (50 s) was used for each light step of RLCs so that N-SLCs differed from RLCs only by the way the amount of light was delivered, i.e., a light dose accumulation for RLC. The experimental results showed the difference between the two photobiological response curves. In the lower range of PFDs, RLCs exhibited a larger value of α; in this light-limited part of the response curve the incremental increase of PFDs limited the development of NPQ and resulted in a better optimization of electron transport rate for RLC. In the higher range of PFDs, the trend was reversed and the RLC showed a lower value of rETR_max than the N-SLC did; this is attributed to the light dose accumulation which likely led to a more efficient dispersion of energy, as illustrated by a higher non-photochemical quenching (NPQ). In conclusion, these results confirm that parameters derived from both methods differ in their value and do not bear the same physiological information.     

Growth response of an early successional assemblage of coralline algae and benthic diatoms to ocean acidification

The sustained absorption of anthropogenically released atmospheric CO₂ by the oceans is modifying seawater carbonate chemistry, a process termed ocean acidification (OA). By the year 2100, the worst case scenario is a decline in the average oceanic surface seawater pH by 0.3 units to 7.75. The changing seawater carbonate chemistry is predicted to negatively affect many marine species, particularly calcifying organisms such as coralline algae, while species such as diatoms and fleshy seaweed are predicted to be little affected or may even benefit from OA. It has been hypothesized in previous work that the direct negative effects imposed on coralline algae, and the direct positive effects on fleshy seaweeds and diatoms under a future high CO₂ ocean could result in a reduced ability of corallines to compete with diatoms and fleshy seaweed for space in the future. In a 6-week laboratory experiment, we examined the effect of pH 7.60 (pH predicted to occur due to ocean acidification just beyond the year 2100) compared to pH 8.05 (present day) on the lateral growth rates of an early successional, cold-temperate species assemblage dominated by crustose coralline algae and benthic diatoms. Crustose coralline algae and benthic diatoms maintained positive growth rates in both pH treatments. The growth rates of coralline algae were three times lower at pH 7.60, and a non-significant decline in diatom growth meant that proportions of the two functional groups remained similar over the course of the experiment. Our results do not support our hypothesis that benthic diatoms will outcompete crustose coralline algae under future pH conditions. However, while crustose coralline algae were able to maintain their presence in this benthic rocky reef species assemblage, the reduced growth rates suggest that they will be less capable of recolonizing after disturbance events, which could result in reduced coralline cover under OA conditions.     

Diurnal variation in chlorophyll fluorescence of Thalassia testudinum seedlings in response to controlled salinity and light conditions

Diurnal variability in chlorophyll fluorescence caused by dynamic irradiance conditions is an important issue when using pulse amplitude modulation fluorometry to measure physiological conditions of plants at the landscape scale. We examined the use of slopes and y-intercepts of diurnal effective photochemical efficiency of photosystem II (PSII) (ΔF/F _m′) versus photosynthetically active radiation (PAR) regressions in addition to direct measurements of maximum photochemical efficiencies of PSII (F _v/F _m) values to assess physiological status of Thalassia testudinum seedlings in a controlled mesocosm study. Seedlings were exposed to two light treatments (full sun and 50–70 % light reduction) and three salinity treatments (20, 35, and 50). Measurements were taken at 0600, 0900, 1200, 1500, 1800, and 2100 hours in order to assess the diurnal variation in photochemical efficiency of PSII and PAR, with measurements at 2100 providing F _v/F _m. Results indicated significant effects of light and salinity on regression y-intercepts and measured F _v/F _m values. Shaded seedlings had higher values for both parameters, suggesting low-light acclimation. The highest salinity treatment resulted in significant reductions for both parameters, suggesting stress. Stress was also indicated by significant reductions in both seedling leaf growth and mean differences between seedling leaves and media osmolalities in the hypersaline treatments (152.0 ± 26.4 vs. 630 ± 40.2 mmol kg^?1 for the control treatments). Slopes of ΔF/F _m′ versus PAR significantly differed with varying light treatments, with full sun seedlings exhibiting shallower slopes than shaded seedlings, indicating higher efficiency of dissipation of excess energy (photoprotection). These experimental results confirm field data suggesting that diurnal ΔF/F _m′ versus PAR regressions are responsive to changes in the physiological status of T. testudinum and that the y-intercepts of diurnal regressions may be used as a proxy for F _v/F _m.     

Pools of chlorophyll and live planktonic diatoms in aphotic marine sediments

Chlorophyll a and numbers of live pelagic diatoms were recorded from sediment depth profiles at 11 stations in the oligotrophic Øresund, Denmark, in late-June. Extraction efficiency of chlorophyll a analysed fluorometrically did not differ significantly between paired samples of frozen-thawed and fresh sediment. The depth profiles of chlorophyll a could be explained by a diagenetic model involving two different chlorophyll pools: one reactive pool declining exponentially with core depth, and one non-reactive pool, of about 1 µg Chl ml^-1 wet sediment, being constant with depth. The number of live diatoms, quantified by the dilution-extinction method, and expressed in terms of most probable number (MPN), declined from an average of about 300,000 g^-1 in the surface sediment to zero values at a depth of 13 cm. The number of live cells was significantly correlated with the sediment chlorophyll a, and the profiles of live cells as well as reactive chlorophyll followed the same exponential decline with core depth, suggesting that the main source of chlorophyll in the sediment was live pelagic diatoms. Taxonomic composition of diatoms in the sediment, dominated by the pelagic genera Chaetoceros, Thalassiosira and Skeletonema, matched the species composition in the water column 3 months earlier during the spring bloom. Regular recordings of the phytoplankton community in the water column showed that only these specific bloom species could be the source of the sediment content of diatoms and chlorophyll a. Further, the ratios between live cells and chlorophyll a were similar in the sediment and in the spring bloom. A conservative estimate of depth-integrated pools of diatoms in the sediment suggested that about 44% of the total phytoplankton biomass during the spring bloom was still present as live cells in the sediment after 3 months. This indicates that the spring bloom input to the sediment is not degraded immediately by the benthic fauna.     

Quantitative evidence concerning the stabilization of sediments by marine benthic diatoms

Six species of benthic diatoms and a natural benthic diatom community were cultured in flasks on a variety of sediments. Diatom species which secreted large quantities of mucilage were effective sediment stabilizers. These mucilage-secreting species significantly reduced resuspension and retarded laminar flow of the sediments when the culture flasks were agitated. Diatom species which secreted little or no mucilage were not effective sediment stabilizers. These non-mucilage-secreting species did not significantly effect resuspension or laminar flow of the sediments when the culture flasks were agitated. A sediment stabilizing mechanism based on the secretion of mucilage by pennate benthic diatoms is proposed. The effect such a process may have on distributional patterns of benthic invertebrates in areas where extensive diatom or other microalgal films occur is discussed.This work was supported by the Belle W. Baruch Foundation, and is Contribution No. 68 of the Belle W. Baruch Institute for Marine Biology and Coastal Research.     

Influence of light and temperature on the photosynthetic rate of marine benthic diatoms

The influence of light and temperature on photosynthetic rate as measured by C¹⁴O₂-fixation of marine benthic diatoms was investigated, using both intact sediment samples (Marshall et al., 1973) and suspensions of diatoms harvested by a lenstissue technique (Eaton and Moss, 1966). After C¹⁴-incubation, sediment samples were filtered, burned in a sample oxidizer, and their activity determined in a liquid scintillation counter. Photosynthetic rate of mixed field populations is saturated by a light intensity of approximately 10,000 lux; at still higher light intensities no photoinhibition was found. In contrast to the mixed field populations, unialgal cultures of the benthic diatom Amphiprora alata Kütz. exhibited strong photoinhibition at higher light intensities (10,000 to 60,000 lux). Within a range of 4° to 20°C, the photosynthetic rate increased about 10%/C^o. No differences in photosynthetic pattern were observed between epipelic and epipsammic species.     

Influence of light and temperature on the growth rate of estuarine benthic diatoms in culture

Four species of estuarine benthic diatoms: Amphiprora c. f. paludosa W. Smith, Nitzschia c. f. dissipata (Kützing) Grunow, Navicula arenaria Donkin, and Nitzschia sigma (Kützing) W. Smith were grown in unialgal cultures. The growth rates of the diatoms were determined as the rate of increase of the chlorophyll a content of the cultures. The diatoms were cultured at different combinations of temperture, daylength, and quantum irradiance. The highest growth rates of Navicula arenaria occurred at 16° to 20°C; the other 3 species had their optimum at 25°C or higher. The small-celled species had higher growth rates at their optimum temperature, but at lower temperatures the growth rates of all 4 species became very similar. The minimum daily quantum irradiance that could effect light-saturated growth at 12° and 20°C ranged from 2.5 to 5.0 E.m^-2.day^-1. At 12°C, two species had their highest growth rates under an 8 h daily photoperiod. At 20°C, the three species tested all had highest growth rates under 16 h daily photoperiod. The growth response of the benthic diatoms is comparable to that of several cultures of planktonic diatoms, as described in the literature. The influence of temperature and quantum irradiance on the diatoms in the present investigation was comparable to the influence of temperature and light intensity on the ¹⁴C-fixation of marine benthic diatoms (Colijn and van Buurt, 1975).     

Constitutional tolerance and chlorophyll fluorescence of Boehmeria nivea L in response to the antimony (Sb) and arsenic (As) co-contamination

The metal tolerance and metabolic changes in Boehmeria nivea colonized in mining areas are not well known. A hydroponic experiment was conducted to evaluate the impact of antimony (Sb)+arsenic (As) in following combinations (control (no metal), 20+0, 10+10, 40+0, 20+20, and 40+40 mg/L) on phytotoxicity, metal tolerance index (MTI), and chlorophyll fluorescence in B. nivea. This constitutes an initial investigation of metal tolerance and chlorophyll fluorescence in Sb and Sb+As contaminated B. nivea. The high Sb+As 40+40 mg/L produced significant phytotoxicity and MTI in the plant. Progressive higher Sb and Sb+As levels resulted in decreased chlorophyll fluorescence of B. nivea. Exposure to intermediate and high Sb+As levels induced damage in the photosynthesis apparatus of the plant.     

Chlorophyll a fluorescence in marine centric diatoms: Responses of chloroplasts to light and nutrient stress

Observations at sea of large variations in the cellular fluorescence of phytoplankton prompted a study of the fluorescence responses in marine diatoms to light and nutrient stress. When older cultures of Lauderia borealis were exposed to intense light, the in vivo fluorescence of chlorophyll a declined within the first 2 min of exposure. This initial response to light stress appeared to be correlated with a contraction of the chloroplasts. Continued exposure led to a second decline in fluorescence, which required 30 to 60 min for completion. A movement of chloroplasts to the valvar ends of the cell caused this secondary response. Both the contraction and intracellular movement of chloroplasts appeared to be related to both photoinhibition of photosynthesis and diel fluctuations in cellular fluorescence. An investigation of continuous cultures of Cyclotella nana showed that in vivo chlorophyll a fluoresced more strongly in nitrogen-starved cells than in enriched ones. Photoinhibition of cellular fluorescence also increased with the cell's state of nitrogen deficiency.     

Tolerance of estuarine benthic diatoms to high concentrations of ammonia,nitrite ion,nitrate ion and orthophosphate

A carbon-14 assimilation method was used to determine action spectra and photosynthesis versus irradiance (P versus I) curves of natural populations of phytoplankton and zooxanthellae from a coral reef fringing Lizard Island in the Australian Barrier Reef. The action spectra were related to the phytoplankton species composition. The curves showed shade adaptation in phytoplankton from deeper waters and in the zooxanthellae. Rates of photosynthesis of zooxanthellae were shown to be highly but variably dependent on their host organisms. Photosynthetic production by zooxanthellae was about 0.9 gC m^-2 day^-1, which is about three times higher than phytoplankton production in the waters close to the reef.     

Quantitative separation of benthic diatoms from sediments using density gradient centrifugation in the colloidal silica Ludox-TM

A quantitative density separation method for benthic diatoms is described in detail. Both epipelic and epipsammic diatoms have been separated from inorganic sediment components including empty diatom frustules. The method is based on differences in specific weight, using medium speed centrifugation on a step gradient of the colloidal silica Ludox-TM. Results obtained with this new method have been compared with those obtained by the lens tissue method. For the density method, applied to samples preserved with a mixture of acrolein, glutaraldehyde and tannic acid, a mean recovery of 82.3% chlorophyll a was found. In the lens tissue harvest-experiments, mean chlorophyll a harvests ranged from 29.1 to 53.2%. Moreover, the standard deviation was several times larger for the lens tissue harvest experiments than for the acrolein series processed by the density method. With the density method, sand fractions were removed entirely, while the clay fractions were removed almost completely. The remaining organic carbon (detritus), with a mean value of 42.3% of the original sample, does not interfere with diatom identification and counting, since all organic matter can be fully oxidized. The density method was found to be applicable to widely different kinds of estuarine sediment and varying quantities of benthic diatom biomass present in the samples. The method can also be used for some other algal groups such as Euglenophyceae and Cyanophyceae.Publication No. 19 of the project Biological Research in the Ems-Dollard Estuary.Communicated by O. Kinne, Hamburg     

Relevance of wound-activated compounds produced by diatoms as toxins and infochemicals for benthic invertebrates

Plants evolve the production of wound-activated compounds (WACs) to reduce grazing pressure. In addition, several plant-produced WACs are recognized by various invertebrates, playing the role of infochemicals. Due to co-evolutionary processes, some invertebrates recognize plant infochemicals and use them to identify possible prey, detect the presence of predators or identify algae containing various classes of toxic metabolites. Different metabolites present in the same algae can play the role of toxins, infochemicals or both simultaneously. We investigated the infochemical activity of compounds extracted from three diatoms epiphytes of the seagrass Posidonia oceanica, by conducting choice experiments on invertebrates living in the same community or in close proximity. Furthermore, the specific toxicity of the extracts obtained from the same algae was tested on sea urchin embryos using a standard bioassay procedure, to detect the presence of toxins. The comparison of the two effects demonstrated that invertebrates are subjected to diatom wound-activated toxicants when these algae are not associated with their own habitat, but they are able to recognize volatile infochemicals derived from diatoms associated with their habitats. The specific toxicity of WACs was shown to be inversely correlated to the perceptive ability of invertebrates towards volatile compounds liberated by the same algae. Hence, when the recognition of specific algae by a given invertebrate species evolves, their detrimental effects on the receiving organism may be lost.     

Differential response of benthic macrofauna and meiofauna to hypoxia in the Gullmar Fjord basin

The response of benthic macro-and meiofauna to severe hypoxia was studied in the deep basis (115 m) of Gullmar Fjord, western Sweden. Abundances and bionasses of the faunal taxa, the redox-potential of the sediment, and the temperature, salinity and dissolved oxygen in the bottom water were recorded over the period 1977 to 1981. In the winter of 1979/80, when a depressed oxygen level of 0.21 ml l^-1 was recorded, the macrofaunal component of the fauna disappeared. The ensuing recolonization, with an initial peak of opportunistic capitellid polychaetes, proceeded slowly and the pre-collapse community was not reestablished within 1 1/2 yr after the hypoxia. In contrast, the permanent meiofauna exhibited no clear signs of being affected by the hypoxia. In the temporary meiofauna, polychaetes seemed to be negatively affected. The finding of a differential response of macro-and meiofauna confirms previous studies which indicate that macrofauna in general is more sensitive than meiofauna to low oxygen concentrations.     

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.     

Sinking rate response to depletion of nitrate,phosphate and silicate in four marine diatoms

The effect of nutrient (N, P, and Si) depletion on sinking rates was studied for two small (Skeletonema costatum (Grev.) Cleve and Chaetoceros gracile Schütt) and two large [Ditylum brightwellii (West) Grun and Coscinodiscus wailesii (Gran et Angst)] centric diatoms obtained from stock cultures. Each diatom was examined under conditions of (1) nutrient repletion (=log growth phase), (2) nutrient depletion (48 h without a given substrate), and (3) recovery (24h after addition of limiting substrate to nutrient-deplete populations). All nutrient-replete cultures displayed low sinking rates despite large differences in cell size. In nutrient-deplete populations, sinking rate was related to the kind of nutrient depleted and varied among species. Silicate depletion elicited by far the greatest increase in sinking rates in all 4 species, indicating that biochemical aspects of silicon metabolism are more important to buoyancy regulation than density-related variations in the amount of silicon per cell. Since N- and P-depletion caused lower sinking rates in 3 of the species, this observation calls for re-evaluation of the axiom that nutrient depletion necessarily causes increased sinking rates. The exception was Coscinodiscus wailesii, which sank faster under all types of nutrient limitation. In most cases, sinking rates typical of log-phase cultures were not regained within 24 h after the addition of limiting nutrient to nutrient-depleted populations. Ultimately, the length of the recovery period may be useful in identifying the metabolic processes responsible for buoyancy regulation in actively growing cells.     

Vertical migratory rhythms of benthic diatoms in a tropical intertidal sand flat: influence of irradiance and tides

Vertical migratory behavior of benthic diatoms is one of the adaptive strategies employed for a life in intertidal habitats. Irradiance and tides are considered to be the key factors governing vertical migration. Experiments were carried out to determine the influence of these factors in a tropical intertidal sand flat. Rising to the sediment surface for fulfilment of their light requirements for photosynthesis was the first priority. If not fulfilled during the low-tide exposure, diatoms could withstand the tidal effects and stay up at the surface even during the high-tide coverage. In the laboratory experiments, where the effects of tides were removed, the endogenous clock continued to operate in a similar fashion to that in the field when under 12-h light:12-h dark conditions, whereas continuous darkness induced a tidal rhythm. In continuous light, diatoms preferred to stay up longer than was observed in field. The above-mentioned observations reveal that irradiance has a stronger effect than tides in controlling/regulating microscale migrations in benthic diatoms. In addition, temporal differences in the irradiance and the resulting changes in diatom migration can have implications for littoral primary productivity.Communicated by O. Kinne, Oldendorf/Luhe     

Growth and competition of the marine diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. II. Light limitation

The marine diatoms Phaeodactylum tricornutum (Bohlin) and Thalassiosira pseudonana (Hasle and Heimdal) were grown under both continous illumination and a 14 h light: 10 h dark cycle at light intensities ranging from 1.53×10^-4 to 2.95×10^-1 ly min^-1. Under both photoperiods, T. pseudonana exhibited higher division rates than P. tricornutum at high light intensities, but the reverse was true at all light intensities <3×10^-3 ly min^-1. Comparison of these results with available data on light-limited growth of other planktonic algae suggests that P. tricornutum may be unusually efficient at maintaining its cell division rate at low light intensity. This efficiency may contribute substantially to its success in turbid, nutrient-enriched mass algal culture systems, the only environments in which it is known to attain great numbers.Contribution No. 4086 from the Woods Hole Oceanographic Institution.     

Chlorophyll fluorescence as a proxy for microphytobenthic biomass: alternatives to the current methodology

Pulse amplitude modulated (PAM) fluorescence has been used as a proxy of microphytobenthic biomass after a dark adaptation period of 15 min to stabilise the minimum fluorescence yield (F _o ¹⁵). This methodology was investigated for in situ migratory and ex situ engineered non-migratory biofilms, comparing dark adaptation to low (5% ambient) and far-red light treatments over different emersion periods. Far-red and low light reduced potential errors resulting from light history effects, by reversal of non-photochemical quenching after 5 min of treatment, compared to over 10 min required by conventional dark adaptation. An in situ decline of minimum fluorescence yield over 15 min was observed during the dark adaptation for migratory biofilms, but was not observed in the non-migratory biofilms indicating that the major cause of decline was downward vertical migration of cells into the sediment. This pattern occurred in far-red light after 10 min, but not for the low light treatment, indicating that low light maintained the biomass at the surface of the sediment. It is therefore concluded that low light treatment is a better option than conventional dark adaptation for the measurement of minimum fluorescence as a proxy of microphytobenthic biomass.