Jacco C. Kromkamp

The use of variable fluorescence measurements in aquatic ecosystems: differences between multiple and single turnover measuring protocols and suggested terminology

In this review, we briefly describe the two main techniques used to measure variable fluorescence in the aquatic environment, and show how the parameters derived from this technique can be used to estimate the rate of photosynthesis. The methods estimate the photochemical efficiency of photosystem II from ratios of fluorescence levels. Flashes of light that are transiently saturating for photochemistry (i.e. they are sufficiently bright to close all PSII reaction centres) are used to obtain the maximum fluorescence level. The type of saturating flash differs between methods. In one approach, single turnover (ST) flashes are applied. This allows only one charge separation during the flash and reduces only the primary acceptor of PS II, raising fluorescence to a level Fm(ST) . In a second approach the flashes are multiple turnover (MT), which allow repeated charge separation processes until all electron acceptors of PS II are reduced. A relaxation of quenching is induced by the longer flash, and this raises the maximum fluorescence to a higher level, Fm(MT) Application of the different approaches to an algal sample will result in differing Fm values and, as a result, different values for the photochemical efficiency of PS II, with the MT method giving higher values than ST. Several designs of equipment, based on MT or ST techniques, are available for use with phytoplankton or benthic algae. Both techniques measure variable fluorescence, but there are a number of important differences in the methods used to calculate photosynthetic rates. In our view, this necessitates the use of a different terminology in order to avoid confusion, until the underlying physiological differences are resolved. An example is given showing that combining terminology from the different approaches will result in calculation of erroneous photosynthetic electron transport rates.

Uncoupling of silicon compared with carbon and nitrogen metabolisms and the role of the cell cycle in continuous cultures of Thalassiosira pseudonana (Bacillariophyceae) under light, nitrogen, and phosphorus control

The elemental composition and the cell cycle stages of the marine diatom Thalassiosira pseudonana Hasle and Heimdal were studied in continuous cultures over a range of different light‐ (E), nitrogen‐ (N), and phosphorus‐ (P) limited growth rates. In all growth conditions investigated, the decrease in the growth rate was linked with a higher relative contribution of the G2+M phase. The other phases of the cell cycle, G1 and S, showed different patterns, depending on the type of limitation. All experiments showed a highly significant increase in the amount of biogenic silica per cell and per cell surface with decreasing growth rates. At low growth rates, the G2+M elongation allowed an increase of the silicification of the cells. This pattern could be explained by the major uptake of silicon during the G2+M phase and by the independence of this process on the requirements of the other elements. This was illustrated by the elemental ratios Si/C and Si/N that increased from 2‐ to 6‐fold, depending of the type of limitation, whereas the C/N ratio decreased by 10% (E limitation) or increased by 50% (P limitation). The variations of the ratios clearly demonstrate the uncoupling of the Si metabolism compared with the C and N metabolisms. This uncoupling enabled us to explain that in any of the growth condition investigated, the silicification of the cells increased at low growth rates, whereas carbon and nitrogen cellular content are differently regulated, depending of the growth conditions.

Bacterial and phytoplankton production in the maximum turbidity zone of three European estuaries: the Elbe, Westerschelde and Gironde

Abstract Biomass and production of phytoplankton and heterotrophic bacteria in spring are presented for three turbid European estuaries, the Elbe (Germany), the Westerschelde (The Netherlands) and the Gironde (France), with emphasis on the effect of turbidity on microbial community densities and activities. Total suspended matter (TSM) concentrations were highest in the Gironde estuary and lowest in the Elbe estuary. Maximum concentrations were found in the maximum turbidity zone (MTZ). Both primary production (PP) and bacterial production (BP) showed a longitudinal gradient with lowest PP and highest BP in the MTZ. Production rates of both phytoplankton and bacterioplankton were lowest in the Gironde and highest in the Westerschelde. PP was positively correlated with the depth of the euphotic zone while BP was positively correlated with TSM and particulate organic carbon. The POC/TSM-ratio, which is related to the degradability of organic carbon, was differed between the three estuaries and was highest in the Westerschelde. The ratio BP:PP was generally very high (>1), and maximal in the MTZ (>4), illustrating the heterotrophic nature of the estuarine ecosystems. Due to the extremely high turbidity in the Gironde, the contribution of bacterial carbon to total microbial biomass (bacteria+algae) was >50%. We conclude that the MTZ has a pronounced impact on the structure and functioning of the microbial community leading to an increased importance of heterotrophic processes and increased degradation of organic material.

Influence of temperature on the relationship between oxygen- and fluorescence-based estimates of photosynthetic parameters in a marine benthic diatom (Cylindrotheca closterium)

In this paper we investigate the temperature sensitivity of the photosynthetic process of the benthic diatom Cylindrotheca closterium grown in light-limited turbidostat cultures at two different growth rates. Photosynthesis was measured as the rate of oxygen evolution and as the photosystem II (PSII) electron transport rate (ETR). The photosynthetic efficiency (a), as measured by both methods, was rather insensitive to temperature, and decreased significantly only at the extreme temperatures used (5 and 35°C). The maximum PSII quantum efficiency (Fv/Fm) showed a small but significant trend of reduction with increasing temperature. However, the maximum rate of photosynthesis (PB max and ETRmax) was extremely temperature sensitive. The effect of temperature on the relationship between PB and ETR was limited to the most extreme temperatures investigated; deviations from linearity were most extreme at 5°C and different conversion factors were observed at 5 and 35°C. A short-term change in temperature (10–30°C), as might be experienced during emersion on a European tidal flat, will not significantly affect the relationship between PB and ETR. However, care should be taken when using a single conversion factor between PB and ETR at the extremes of the temperature range. We have also shown that algal absorption measurements are important for correct calculation of ETR. The facts that different species seem to have different conversion factors and that changing environmental conditions will affect the absorption capacity and growth rate of the microphytobenthos (MPB) community suggest that it is wise to perform further calibrations of the relationship in the field before use in primary production modelling. Variable fluorescence measurements are quick and non-invasive and, with knowledge of the absorption properties of the MPB community, allow the quantification of photosynthetic parameters across large areas. Hence they are potentially useful for improving our estimates of ecosystem scale primary production.

Formation and functional significance of storage products in cyanobacteria

Abstract The most common storage products of cyanobacteria are polyphosphate as a phosphorus storage compound, cyanophycin or phycobilin protein pigment as nitrogen storage products, and glycogen as a storage product of both carbon and energy. Nutrient uptake kinetics are regulated by the storage pools, and the patterns of regulation have a feedback effect on the amount of accumulated nutrient in the cells. Besides having a storage function the nutrient storage products are likely to act as metabolic sinks during conditions of energy stress. Regulation of storage products is especially strict in light‐limited cultures. By increasing the rate of polysaccharide formation during growth with short photoperiods, cyanobacteria are able to sustain relatively high growth rates. This effect is enhanced by keeping respiratory losses very low.

Estimating phytoplankton primary production in Lake IJsselmeer (The Netherlands) using variable fluorescence (PAM-FRRF) and C-uptake techniques

In this paper we compare measured rates of C-fixation obtained using in situ deployment of a fast repetition rate fluorometer (FRRF) applying two different calculation protocols and a waterPAM fluorometer. The measurements were carried out in the large, shallow Lake IJsselmeer (The Netherlands). Turbidity appears to be the most important factor in explaining photosynthetic parameters. Non-photochemical quenching (NPQ) in the antenna was observed, and the degree of NPQ was positively correlated with the irradiance and mixing conditions. However, NPQ did not affect the functional cross-section. Conversion of photosynthetic electron transport rates (ETR) into rates of carbon fixation requires knowledge of two or three parameters (depending on the protocol and instrument used) to calculate C-fixation from ETR: the electron yield (Φe, i.e. the reciprocal of the quantum requirement of PSII), the photosynthetic quotient (PQ) and the number of PSII (nPSII). Using normally assumed values for these three parameters resulted in overestimation of the measured rate of C-fixation but, in all cases, ETR was linearly related to it. The degree of overestimation was rather constant, despite considerable changes in phytoplankton composition. Spectral correction of fluorescence data caused a reduction of 30% or an increase of 44% in the estimated C-fixation, depending how C-fixation was estimated. We describe a method that allows estimation of the Φe/PQ ratio based on a comparison of the quantum efficiencies for C-fixation and PSII and show that, with a single and realistic value for this ratio, primary production could be accurately predicted at different times of the year.

REGULATION OF GAS VESICLE CONTENT AND BUOYANCY IN LIGHT-OR PHOSPHATE-LIMITED CULTURES OF APHANIZOMENON FLOS-AQUAE (CYANOPHYTA)1

Measurements of the gas vesicle space in steady‐state light or phosphate‐limited cultures of Aphanizomenon flos‐aquae Ralfs, strain 7905 showed that gas vesicle content decreased as energy‐limited growth rate increased hut was the same at several phosphate‐limited growth rates. Upon a decrease in growth irradiance, gas vesicle content did increase in phosphate‐limited cultures, hut the cultures remained nonbuoyant as long as P was limiting. Buoyant, energy‐limited cultures lost their buoyancy in less than 2 h when exposed to higher irradiances. The primary mechanism for buoyancy loss was the accumulation of polysaccharide as ballast. Collapse of gas vesicles by turgor pressure played a minor role in the loss of buoyancy. When cultures were exposed to higher irradiances, cells continued to synthesize gas vesicles at the same rate as before the shift for at least 1 generation time. The amount of ballast required to make individual filaments in the population sink varied 4‐fold. This variation appears to be due to differences in gas vesicle content among individual filaments.

Relationship of intertidal surface sediment chlorophyll concentration to hyperspectral reflectance and chlorophyll fluorescence

Estimating biomass of microphytobenthos (MPB) on intertidal mud flats is extremely difficult due to their patchy occurrence, especially at the scale of an entire mud flat. We tested two optical approaches that can be applied in situ: spectral reflectance and chlorophyll fluorescence. These two approaches were applied in 4 European estuaries with different sediment characteristics. At each site, paired replicate measurements of hyperspectral reflectance, chlorophyll fluorescence (after 15 min dark adaptation, Fo15), sediment water content, and chlorophylla concentrations were taken (including breakdown products: [chla+phaeo]). Sediments were further characterized by grain size and organic content analysis. The spectral signatures of tidal flats dominated by benthic microalgae, mainly diatoms, could be easily distinguished from sites dominated by macrophytes; we present a 3 waveband algorithm that can be used to detect the presence of macrophytes. The normalized difference vegetation index (NDVI) was found to be most strongly correlated to sediment [chla+phaeo], except for the predominantly sandy Sylt stations. Fo15 was also significantly correlated to sediment [chla+phaeo] in all but one grid (Sylt grid A). Our results suggest that the functional relationships (i.e., the slopes) between NDVI or fluorescence and [chla+ phaeo] were not significantly different in the muddier grids, although the intercepts could differ significantly, especially for Fo15. This suggests a mismatch of the optical depth seen by the reflectometer or fluorometer and the depth sampled for pigment analysis. NDVI appears to be a robust proxy for sediment [chla+phaeo] and can be used to quantify MPB biomass in muddy sediments of mid latitude estuaries.

Effect of short-term variation in irradiance on light harvesting and photosynthesis of the marine diatom Skeletonema costatum: a laboratory study simulating vertical mixing

SUMMARY: A laboratory study was conducted into the physiology of Skeletonema costatum grown under a simple sinusoidal and a fluctuating light regime. The latter simulated a light regime similar to that which could result from the vertical mixing caused by Langmuir circulation. It was shown that the culture simulating vertical mixing reacted by decreasing the photosynthetic unit (PSU) size and increasing the number of PSUs, and hence optimized the rate of maximal photosynthesis at high, saturating irradiances. This culture also showed some change in photosynthetic parameters during the light period, which was especially pronounced during the shift from a low to a higher irradiance. The effect of this on estimates of primary production in a water column is discussed. Further, it is speculated that the assimilation number is regulated by the maximum light intensity experienced during the day rather than the total daily light dose, because only the culture submitted to a fluctuating light regime showed a real change in the maximum rate of photosynthesis (P B max) upon transfer to higher light levels.

Buoyancy regulation in a strain of Aphanizomenon flos-aquae (Cyanophyceae): the importance of carbohydrate accumulation and gas vesicle collapse

Buoyancy regulation in light-limited continuous cultures of Aphanizomenon flos-aquae was studied. Gas vesicle collapse did not occur during growth under a light-dark cycle. Only cultures with growth rates less than 20% of the maximal growth rate were positively buoyant. The loss of buoyancy at higher growth rates was due to a lower gas vesicle content in the cells. Carbohydrate accumulation was the main factor which caused buoyancy loss if the cells were shifted from low to high photon flux densities. In these experiments turgor-induced gas vesicle collapse occurred only in cultures adapted to long light periods, and several hours after the cells had lost buoyancy due to ballast increase. The results are discussed in relation to adaptation patterns in photosynthesis and carbon metabolism caused by intermittent light.