Ladislav Nedbal

Influence of high frequency light/dark fluctuations on photosynthetic characteristics of microalgae photoacclimated to different light intensities and implications for mass algal cultivation

Oxygen evolution from aScenedesmus obliquus dominated outdoor culture was followed in a small volume chamber, irradiated either by continuous white light or under light/dark frequencies between 0.05 to 5000 Hz, using arrays of ‘high intensity’ red light emitting diodes (LEDs). By placing neutral density filters in the path of the white light, light saturation curves of the oxygen evolution (P/I curves) were measured using diluted aliquots of algal cultures. The results clearly showed that photosynthetic rates increased exponentially with increasing light/dark frequencies, that a longer dark period in relation to the light period does not necessarily lead to higher photosynthetic rates (efficiencies), and that algae do not acclimate to a specific light/dark frequency. One of the most important factors that influenced photosynthetic rates, either under continuous illumination or intermittent, was whether the algae were dark or light acclimated. Low light/dark frequencies were perceived by the algae as low light conditions, whilst the opposite was true for high frequencies. The light utilisation efficiency in a fluctuating light/dark environment depended on the acclimated state of the algae, the specific frequency of the fluctuations and the duration of the exposure. Since the frequencies determined the ‘perceived’ quantities of light, dark reactions played an important role in determining the average photosynthetic efficiencies. These results have important implications for algal biotechnology.

Kinetic imaging of chlorophyll fluorescence using modulated light

Fluorometers that measure the kinetics of chlorophyll fluorescence have become invaluable tools for determining the photosynthetic performance of plants. Many of these instruments use high frequency modulated light to measure the rate, efficiency and regulation of photosynthesis. The technique is non-invasive and is effective under diverse environmental conditions. Recently, imaging fluorometers have been introduced that reveal variability in photosynthesis over the surface of a leaf or between individual plants. Most imaging instruments depend on continuous light or low frequency modulated light for fluorescence excitation, which imposes serious limitations on measurements of the fluorescence parameters, especially the minimum fluorescence (F0) and variable fluorescence (FV). Here, we describe a new instrument that combines the advantage of high frequency modulated light with two-dimensional imaging of chlorophyll fluorescence. The fluorometer produces dynamic images of chlorophyll fluorescence from leaves or plants, providing accurate mapping of F0 and FV, and non-photochemical quenching. A significant feature of the instrument is that it can record fluorescence images of leaves in daylight under field conditions.

Scientific and technical challenges in remote sensing of plant canopy reflectance and fluorescence

State-of-the-art optical remote sensing of vegetation canopies is reviewed here to stimulate support from laboratory and field plant research. This overview of recent satellite spectral sensors and the methods used to retrieve remotely quantitative biophysical and biochemical characteristics of vegetation canopies shows that there have been substantial advances in optical remote sensing over the past few decades. Nevertheless, adaptation and transfer of currently available fluorometric methods aboard air- and space-borne platforms can help to eliminate errors and uncertainties in recent remote sensing data interpretation. With this perspective, red and blue-green fluorescence emission as measured in the laboratory and field is reviewed. Remotely sensed plant fluorescence signals have the potential to facilitate a better understanding of vegetation photosynthetic dynamics and primary production on a large scale. The review summarizes several scientific challenges that still need to be resolved to achieve operational fluorescence based remote sensing approaches.

Microscopic green algae and cyanobacteria in high-frequency intermittent light

The effects of fluctuations in the irradiance onScenedesmus quadricauda, Chlorella vulgaris andSynechococcus elongatus were studied in dilute cultures using arrays of red light emitting diodes. The growth rate and the rate of photoinhibition were compared using intermittent and equivalent continuous light regimes in small-size (30 ml) bioreactors. The CO2 dependent photosynthetic oxygen evolution rates in the intermittent and continuous light regimes were compared for different light/dark ratios and different mean irradiances. The kinetics of the electron transfer reactions were investigated using a double-modulation fluorometer. The rates of photosynthetic oxygen evolution normalized to equal mean irradiance were lower or equal in the intermittent light compared to the maximum rate found in the equivalent optimal continuous light regime. In contrast, the growth rates in the intermittent light can be higher than the growth rate in the equivalent continuous light. Photoinhibition is presented as an example of a physiological process affecting the growth rate that occurs at different rates in the intermittent and equivalent continuous lights. The difference in the dynamics of the redox state of the plastoquinone pool is proposed to be responsible for the low photoinhibition rates observed in the intermittent light.

Raman Microspectroscopy of Individual Algal Cells: Sensing Unsaturation of Storage Lipids in vivo

Algae are becoming a strategic source of fuels, food, feedstocks, and biologically active compounds. This potential has stimulated the development of innovative analytical methods focused on these microorganisms. Algal lipids are among the most promising potential products for fuels as well as for nutrition. The crucial parameter characterizing the algal lipids is the degree of unsaturation of the constituent fatty acids quantified by the iodine value. Here we demonstrate the capacity of the spatially resolved Raman microspectroscopy to determine the effective iodine value in lipid storage bodies of individual living algal cells. The Raman spectra were collected from three selected algal species immobilized in an agarose gel. Prior to immobilization, the algae were cultivated in the stationary phase inducing an overproduction of lipids. We employed the characteristic peaks in the Raman scattering spectra at 1,656 cm−1 (cis C═C stretching mode) and 1,445 cm−1 (CH2 scissoring mode) as the markers defining the ratio of unsaturated-to-saturated carbon-carbon bonds of the fatty acids in the algal lipids. These spectral features were first quantified for pure fatty acids of known iodine value. The resultant calibration curve was then used to calculate the effective iodine value of storage lipids in the living algal cells from their Raman spectra. We demonstrated that the iodine value differs significantly for the three studied algal species. Our spectroscopic estimations of the iodine value were validated using GC-MS measurements and an excellent agreement was found for the Trachydiscus minutus species. A good agreement was also found with the earlier published data on Botryococcus braunii. Thus, we propose that Raman microspectroscopy can become technique of choice in the rapidly expanding field of algal biotechnology.

A Photobioreactor System for Precision Cultivation of Photoautotrophic Microorganisms and for High-Content Analysis of Suspension Dynamics

Small‐scale photobioreactors for cultivation of photoautotrophic microbes are required for precise characterization of the growth parameters of wild‐type and engineered strains of these organisms, for their screening, and for optimization of culture conditions. Here, we describe the design and use of a flat‐cuvette photobioreactor that allows accurate control of culture irradiance, temperature, pH, and gas composition combined with real‐time monitoring by a built‐in fluorometer and densitometer. The high‐power LED light source generates precise irradiance levels that are programmed by user‐designed protocols. The irradiance, temperature, and gas composition may be static or dynamically modulated, while optical density and pH may be stabilized in turbidostat and pH‐stat modes, respectively. We demonstrate that the instrument is able to detect minute variations of growth caused, for example, by sudden dilution or by circadian rhythms. The sensitivity of the instrument is sufficient to monitor suspension optical density as low as 10−2. This newly designed photobioreactor can significantly contribute to the study and use of photoautotrophic microbes in systems biology and biotechnology. Biotechnol. Bioeng. 2008;100: 902–910.

Flash fluorescence induction: a novel method to study regulation of Photosystem II

Abstract We demonstrate that arrays of light-emitting diodes can be used to generate single-turnover flashes of light that saturate Q A reduction in green algae. The fast version of a double-modulation fluorometer can measure induction during a single-turnover saturating flash (flash fluorescence induction). The method allows the effective antenna size, antenna heterogeneity and connectivity of Photosystem II to be measured without poisoning the organism with herbicide that blocks Q A − reoxidation. Using the same instrument, we have also measured the functional heterogeneity of Photosystem II by the fluorescence transient related to the flash-induced advancement of S-states in the oxygen-evolving complex.

Dual-modulation LED kinetic fluorometer

Abstract The instrument generates, from a combined array of light emitting diodes (LED), non-periodical flashes of variable spectral composition and of user-defined timing, energy and duration. In a standard application, the instrument is used to excite photochemistry in photosynthetically active samples by strong single-turnover flashes and to follow the elicited electron-transfer reactions using weak non-periodic measuring flashes. The energy of the measuring flashes is low so that their contribution to the photochemistry is negligible and, yet, it is sufficient to excite easily detectable fluorescence emission and to allow detection of photochemistry-reflecting absorption changes in the range of A ≈ 10−3. The minimal timing-definition step for a trigger or measure event is 300 ns and the minimal interval between two measurements is 10 μs for each of the two independent input channels. The data are read with 16 bit resolution.

Engineered drought tolerance in tomato plants is reflected in chlorophyll fluorescence emission

Drought stress is one of the most important factors that limit crop productivity worldwide. In order to obtain tomato plants with enhanced drought tolerance, we inserted the transcription factor gene ATHB-7 into the tomato genome. This gene was demonstrated earlier to be up-regulated during drought stress in Arabidopsis thaliana thus acting as a negative regulator of growth. We compared the performance of wild type and transgenic tomato line DTL-20, carrying ATHB-7 gene, under well-irrigated and water limited conditions. We found that transgenic plants had reduced stomatal density and stomatal pore size and exhibited an enhanced resistance to soil water deficit. We used the transgenic plants to investigate the potential of chlorophyll fluorescence to report drought tolerance in a simulated high-throughput screening procedure. Wild type and transgenic tomato plants were exposed to drought stress lasting 18 days. The stress was then terminated by rehydration after which recovery was studied for another 2 days. Plant growth, leaf water potential, and chlorophyll fluorescence were measured during the entire experimental period. We found that water potential in wild type and drought tolerant transgenic plants diverged around day 11 of induced drought stress. The chlorophyll fluorescence parameters: the non-photochemical quenching, effective quantum efficiency of PSII, and the maximum quantum yield of PSII photochemistry yielded a good contrast between wild type and transgenic plants from day 7, day 12, and day 14 of induced stress, respectively. We propose that chlorophyll fluorescence emission reports well on the level of water stress and, thus, can be used to identify elevated drought tolerance in high-throughput screens for selection of resistant genotypes.

Evidence for a role of raffinose in stabilizing photosystem II during freeze–thaw cycles

A role of non-reducing sugars like sucrose and raffinose in the protection of plant cells against damage during freezing has been proposed for many species, but reports on physiological effects are conflicting. Non-aqueous fractionation of mesophyll cell compartments in Arabidopsis thaliana was used to show that sucrose and raffinose accumulate in plastids during low temperatures, pointing to a physiological role in protecting the photosynthetic apparatus. Comparing a previously described raffinose synthase (RS) mutant of A. thaliana with its corresponding wild type, accession Col-0, revealed that a lack of raffinose has no effect on electrolyte leakage from leaf cells after freeze–thaw cycles, supporting that raffinose is not essential for protecting the plasma membrane. However, in situ chlorophyll fluorescence showed that maximum quantum yield of PS II photochemistry (Fv/Fm) and other fluorescence parameters of cold acclimated leaves subjected to freeze–thaw cycles were significantly lower in the raffinose synthase mutant than in the corresponding wild type, indicating that raffinose is involved in stabilizing PS II of cold acclimated leaf cells against damage during freezing.