Vanda Brotas

The use of HPLC pigment analysis to study microphytobenthos communities

The use of HPLC pigment analysis has become a widespread tool for investigating the taxonomic composition of phytoplankton. However, for micropytobenthos, due to the complexity of these communities and the accumulation of vegetal senescent material, the application of this technique presents further problems. Samples of unialgal cultures from representative taxa of microphytobenthos and samples of surface sediment from intertidal mudflats were collected, extracted in methanol and analyzed for photosynthetic pigments in a HPLC system, with a fluorimeter and a photodiode array detector. A total of 11 chlorophylline pigments and 19 carotenoids were identified. Absorbance chromatograms from cultures and natural populations are displayed. Pigment ratios from five species belonging to different taxonomic classes are compared. Pigment composition and diversity of intertidal muddy sites are discussed; the increase of degraded compounds of chlorophyll a and carotenoids along a vertical profile of 5 cm is shown on different sediment types.

Variability in Chlorophyll and Phytoplankton Composition in an Estuarine System

The spatial and temporal variability of phytoplankton abundance (in terms of chlorophyll a and cell number), inorganic nitrogen, suspended particulate matter (SPM), and light availability was determined throughout one year in the Tagus estuary, Portugal. Chlorophyll a concentrations showed a strong seasonal variation with values ranging from 1 to 32 μg l−1(average 5.4 μg l−1). Chlorophyll patterns were unimodal for sites 1, 2, and 3 and bimodal for site 4. Diatoms and cryptophytes were, throughout the year, the dominant groups in this shallow and unstratified estuarine system. Nitrate concentrations were seasonally related to river flow and ammonium concentrations spatially related to sources of sewage input. Lower river inputs and long water residence times during summer initially promoted the accumulation of phytoplankton, but the resulting low dissolved inorganic nitrogen (DIN) concentrations lead to limitation of phytoplankton growth. Chlorophyll a and DIN values obtained in the present study were comparable to those reported 20 years ago for the Tagus estuary.

Oxygen profiles in intertidal sediments of Ria Formosa (S. Portugal)

Microelectrode oxygen profiles were measured in intertidal sediments from Ria Formosa (S. Portugal), a very productive shallow coastal lagoon. Four intertidal sampling sites were selected according to different sediment characteristics. Individual profiles revealed a high degree of lateral variability on a centimeter spatial scale. Nevertheless, consistent differences were observed between oxygen profiles measured in atmosphere-exposed and inundated intertidal sediments: in organically poor sand oxygen-penetration depth varied from 3 mm in inundated cores to more than 7 mm in exposed ones, while in organically rich muddy sand and mud it remained between 0.5–2.0 mm. The oxygen input from inundated to exposed conditions was estimated for each sampling site. Semi-diurnal tidal fluctuation, leading to periodical atmospheric exposure of sediments plays a major role in the oxygenation process of intertidal zones of Ria Formosa.

Monitoring Migration and Measuring Biomass in Benthic Biofilms: The Effects of Dark/far-red Adaptation and Vertical Migration on Fluorescence Measurements.

Pulse modulated fluorescence has increasingly been used as an ecological tool to examine changes in the vertical distribution of microphytobenthic cells within the upper layers of estuarine sediments (most often using the minimum fluorescence yield Fo) as well as to indicate the health of the community (using the maximum PS II quantum efficiency Fv/Fm). However, the practicalities of in situ measurements, often dictates that short dark adaptation periods must be used (∼15 min). The use of far-red light as an alternative to dark adaptation was investigated in natural migratory microphytobenthic biofilms and artificial non-migratory biofilms. Prolonged periods of darkness (∼24 h) were not adequate to achieve ‘true’ measurements of Fo and Fv/Fm, which require complete oxidation of QA and full reversal of non-photochemical quenching (NPQ). In some instances, stable values were only achieved using far-red light. Prolonged exposure to dark/far-red light led to a downwards migration of cells in natural assemblages, as seen by a reduction in both Fo and the maximum fluorescence yield (Fm). In non-migratory biofilms, Fm increased in the dark and far-red treatments, indicating a reversal of NPQ, whereas Fo decreased in far-red light but increased in the dark. It is suggested that far-red light and darkness differentially affected the balance between NPQ reversal and QA oxidation that lead to the measured Fo yield. The use of far-red light as an alternative to dark adaptation is discussed and the implications of short (e.g., 15 min) dark adaptation times used in situ are discussed with reference to the vertical migration of cells within sediment biofilms.

Shifts in the dominance between diatoms and cryptophytes during three late summers in the Bransfield Strait (Antarctic Peninsula)

Recent global warming reduces surface water salinity around the Antarctic Peninsula as a result of the glacial meltwater runoff, which increases the occurrence and abundance of certain phytoplankton groups, such as cryptophytes. The dominance of this particular group over diatoms affects grazers, such as Antarctic krill, which preferentially feed on diatoms. Using three late summer data sets from the Bransfield Strait (2008–2010), we observed variations in the dominant phytoplankton groups determined by HPLC/CHEMTAX pigment analysis and confirmed by microscopy. Results indicate that the dominance of diatoms, particularly in 2008 and 2009, was associated with a deeper upper mixed layer (UML), higher salinity and warmer sea surface temperature. In contrast, cryptophytes, which were dominant in 2010, were associated with a shallower UML, lower salinity and colder sea surface temperatures. The low diatom biomass observed in the summer of 2010 was associated with high nutrient concentration, particularly silicate, and low chlorophyll a (summer monthly average calculated from satellite images). The interannual variability here observed suggests a delayed seasonal succession cycle of phytoplankton in the summer of 2010 associated with a cold summer and a late ice retreat process in the region. This successional delay resulted in a notable decrease of primary producers’ biomass, which is likely to have impacted regional food web interactions. This study demonstrates the susceptibility of the Antarctic phytoplankton community structure to air temperature, which directly influences the timing of ice melting and consequently the magnitude of primary production and succession pattern of phytoplankton groups.

Microphytobenthos primary production of Tagus estuary intertidal flats (Portugal)

The purpose of this study was to measure primary productivity of microphytobenthos of intertidal mudflats of Tagus estuary. Sampling occurred from 1991 to 1992, during low tide of spring waters on 2 stations, each one representative of a typical habitat. Net photosynthesis rates (NP) were measured in undisturbed sediment cores incubated in the laboratory andin situ. The results obtained indicated that NP in the laboratory was similar for the two stations, (mean values of 1.1 and 1.3 mmol O2 m−2 h−1, within a range of 5-fold). A stepwise regression analysis combining biotic and abiotic factors was performed to explain temporal variability, indicating that NP in each site was influenced by different parameters, with the exception of precipitation, which might play an important role in the disruption of sediment-water interface. Photosynthesis-Irradiance curves constructed with the results obtained insitu showed similar photophysiological responses of the two communities. Based on the values of photosynthetic efficiency (α), and of optimal light intensity (lk) measured and on the combination of the photoperiod pattern and the amount of light available for each sampling site, an estimation of total annual productivity is given: 47–178 g C m−2.

Pheophorbide and pheophytin a-like pigments as useful markers for intertidal microphytobenthos grazing by Hydrobia ulvae

Pigment degradation by Hydrobia ulvae, feeding on an intertidal mudflat dominated by diatoms, was studied in the laboratory. HPLC analysis identified significantly higher amounts of pheopigments in grazed sediment and a decrease in the concentrations of major chlorophylls (a and c1+c2) and carotenoides (fucoxanthin, diadinoxanthin, and β-carotene). Grazing by H. ulvae leads to a stronger pheophorbide a accumulation relative to pheophytin a. Dominant chlorophyll a degradation products were the less polar pheophorbide a4 and pheophytin a2. These pigments were identified in very high concentrations in H. ulvae faecal pellets. Although a major disadvantage of the HPLC method is the uncertainty regarding the extent and rate of pigment conversion into colourless products, these data strongly suggest the possible use of pheophorbide a4 and pheophytin a2 as useful markers for intertidal microphytobenthos grazing by H. ulvae.

Taxonomic survey of the microphytobenthic communities of two Tagus estuary mudflats

A taxonomic survey of the microphytobenthic communities was conducted in two intertidal mudflats of the Tagus estuary. These mudflats are located in different littoral zones of the estuary and had distinct sediment characteristics and salinity range. Sampling was performed monthly during low tide over a 14-month-period. Sediment cores were collected and brought to the laboratory, where the microalgae were harvested using the lens-tissue technique under light stimulation. Samples were examined using both light and scanning electron microscopy. The epipelic pennate diatoms dominated the microphytobenthic assemblages on both mudflats but cyanobacteria and euglenophytes were also found. Of the 52 taxa identified, 47 were pennate diatoms, five of which seem to be previously unreported for Portugal. The species more frequently found in the samples were Gyrosigma fasciola, Cylindrotheca cf. signata, Cylindrotheca closterium, Staurophora amphioxys and Navicula gregaria.

In situ measurements of photosynthetic activity and respiration of intertidal benthic microalgal communities undergoing vertical migration

Abstract This paper presents the results of a field study on the short-term variability in photosynthetic activity of inter-tidal microphytobenthos using non-destructive techniques in situ. Photosynthetic rate (measured using oxygen microelectrodes), productive biomass and photosynthetic efficiency (dark-fluorescence and effective quantum efficiency of PSII, respectively, measured by PAM fluorometry) were measured in situ during a low-tide exposure period on an intertidal mudflat in the Tagus Estuary, Portugal. Community photosynthesis displayed large temporal variation. Maximum activity (24.3 mmol O2 m-2 h-1) was found at noon while minimum activity (8.7 mmol O2 m-2 h-1) was found at the end of the exposure period. Productive biomass followed a similar pattern. During the course of the exposure period, productive biomass peaked halfway through, and decreased by 80% towards the end of the exposure period. These changes in productive biomass were followed by significant changes in the species composition/cell size as well as changes in photosynthetic efficiency of the microphytobenthic community in the surface sediment. The obtained results therefore suggest that microphytobenthic community-level photosynthesis is controlled by migratory movements of microalgae and by variations in photosynthetic efficiency. The latter could be induced by changes in the species composition/cell size in the photic zone due to microalgal migrations. It is suggested that the migration pattern and, hence, photosynthesis is linked to changes in salinity, temperature and light, changes that are difficult to simulate accurately in the laboratory. Respiration rates obtained during the day were much higher than those obtained at night, and also displayed a marked hour-to-hour variation. Depth integrated respiration within the photosynthesis zone (0.33 mm) represented 80 to 90% of the respiration in the entire oxic zone, suggesting a tight coupling between photosynthesis and respiration.

Photoacclimation in microphytobenthos and the role of xanthophyll pigments

Estuarine microphytobenthos are frequently exposed to excessively high irradiances. Photoinhibition in microalgae is prevented by various photophysiological responses. We describe here the role of the xanthophyll pigments in photoacclimation. The pigment composition of the microphytobenthos was studied in three European estuaries (Barrow, Ireland; Eden, UK; Tagus, Portugal). Using HPLC-analyses, microscale changes in biomass and pigment composition were monitored over short (hourly) and long (seasonal) time scales. In the Barrow estuary, the biomass of microphytobenthos (measured as chlorophyll a) increased significantly in the top 400–500 µm of the sediment surface within 1 h of emersion; simultaneously, the xanthophyll pool size (diadinoxanthin plus diatoxanthin, dd + dt) almost doubled. A more gradual conversion of dd into dt was observed, with the dt:dd ratio increasing from <0.1 at the start of emersion to >0.3 after 3 h emersion. Similar trends in the dt:dd ratio were observed in the surface sediments of the Eden and the Tagus estuaries. Higher ratios were recorded in the Tagus estuary, which may be explained by higher incident irradiance. In addition, seasonal studies carried out in the Eden and Tagus estuaries showed that the xanthophyll pool size increased by 10% in the summer months. The pool size was highest in the Tagus estuary. Concurrently, high values for the de-epoxidation state were recorded, with values for dt/(dt + dd) > 0.35 recorded in the summer. At the Eden, the ratio never exceeded 0.3. The de-epoxidation state was higher in winter than in summer, which was ascribed to the low winter temperatures. During a vertical migration study, a negative relationship between chlorophyll a and the de-epoxidation state was observed. It is suggested that this relationship originates from ‘micro-migration’ within the biofilm. Migration within the euphotic zone may provide an alternative means for cells to escape photodamage. In this paper, we propose that both xanthophyll cycling and ‘micro’-migration play an important role in photoacclimation and it appears that these processes operate in parallel to regulate the photosynthetic response.