A mid-infrared spectroscopic method was developed for the simultaneous and quantitative determination of total protein, carbohydrate and lipid contents of microalgal cells. Based on a chemometric approach, measured FTIR (Fourier transform infrared) spectra from algal cells were reconstructed by a partial least square algorithm, using the spectra of the reference substances to determine their relative contribution to the overall cell spectrum. From this specific absorption, absolute macromolecular cell composition [pg cell(-1)] can be calculated using calibration curves, which have been validated by independent biochemical methods. The future potential of this method for photosynthesis research is shown by its application to follow time-resolved changes in the cellular composition of microalgae during an illumination period of several hours. We show how the macromolecular composition can be investigated by FTIR spectroscopy methods. This can substantially increase the efficiency of screening processes like bioreactor monitoring and may be beneficial in metabolic engineering of algal cells.

Summary Plant cells harbor two types of endosymbiotic organelle: mitochondria and chloroplasts. As a consequence of endosymbiotic gene transfer, the majority of their proteins are encoded in the nucleus and post‐translationally ‘re’‐imported into the respective target organelle. The corresponding transport signals are usually selective for a single organelle, but several proteins are transported into both the mitochondria and chloroplasts. To estimate the number of proteins with such dual targeting properties in Arabidopsis, we classified the proteins encoded by nuclear genes of endosymbiotic origin according to the respective targeting specificity of their N‐terminal transport signals as predicted by the TargetP software package. Selected examples of the resulting protein classes were subsequently analyzed by transient transformation assays as well as by in organello protein transport experiments. It was found that most proteins with high prediction values for both organelles show dual targeting with both experimental approaches. Unexpectedly, however, dual targeting was even found among those proteins that are predicted to be localized solely in one of the two endosymbiotic organelles. In total, among the 16 candidate proteins analyzed, we identified 10 proteins with dual targeting properties. This unexpectedly high proportion suggests that such transport properties are much more abundant than anticipated.

The performance and methodological limits of the Phyto-PAM chlorophyll fluorometer were investigated with laboratory grown algae cultures and natural phytoplankton from the rivers Saar and Saale. The Phyto-PAM is a 4-wavelength chlorophyll fluorometer with the functional combination of chlorophyll (Chl) estimation and assessment of photosynthetic activity, both differentiated into the main algal groups. The reliability of fluorescence-based Chl estimation strongly depends on the group specific calibration of the instrument and the resulting chlorophyll/fluorescence (Chl/F) ratios in reference algal cultures. A very high reliability of the Chl estimation was obtained in the case of constant Chl/F-ratios. Algae grown at different light intensities showed marked differences in Chl/F-ratios, reflecting differences in pigment composition and Chl a specific absorption (a*). When the Phyto-PAM was calibrated with laboratory grown diatoms, the Chl a in river grown diatoms was underestimated, due a lower content of accessory pigments and stronger pigment packaging. While this aspect presently limits the application of PAM fluorometry in limnology, this limitation may be overcome by future technical progress in the detection of dynamic changes in Chl/F-ratio via fluorescence-based measurements of the functional PS II absorption cross-section. Practically identical Chl/F-ratios were found for the diatom-dominated waters of the rivers Saar and Saale, suggesting that the same instrument calibration parameters may be applied for hydrographically similar surface waters. For this particular case, despite of the present methodological limitations, the potential of PAM fluorometry in limnology could be demonstrated. Light response curves were measured to estimate primary production with a spectrally resolved model in daily courses at two sampling sites. Fluorescence based primary production was closely correlated with measured oxygen evolution rates until midday. In the afternoon, at the water surface the fluorescence approach gave higher rates than the measured oxygen evolution. Possible explanations for the observed differences are discussed.

Chlamydomonas is one of the most well-studied photosynthetic organisms that had important biotechnological potential for future bioproductions of biofuels. However, an energy balance from incident photons to the energy stored in the new biomass is still lacking. In this study, we applied a recently developed system to measure the energy balance for steady state growth of Chlamydomonas reinhardtii grown at pH 6.5, and C. acidophila that was grown at pH 6.5 and 2.6. Energy use efficiency was quantified on the basis of light absorption, photosynthetic quantum yield, photosynthetic and respiratory quotient, and electron partitioning into proteins, carbohydrates and lipids. The results showed that lower growth rates of C. acidophila under both pH conditions were not caused by the differences in the photosynthetic quantum yield or in alternative electron cycling, but rather by differences in the efficiency of light absorption and increased dark respiration. Analysis of the macromolecular composition of the cells during the light phase showed that C. acidophila uses biosynthetic electrons preferentially for carbohydrate synthesis but not for synthesis of lipids. This led to a strong diurnal cycle of the C/N ratio and could explain the higher dark respiration of C. acidophila compared with C. reinhardtii.

The phytoplankton community structure of a hypertrophic lake was quantitatively determined with the aid of flow cytometry. The flow cytometry signals were calibrated to obtain cell-specific information, such as the chl a content and the biovolume per cell. The reliability of this method was tested with laboratory cultures. The results of the phytoplankton structure in a hypertrophic lake with respect to chl distribution in the different algal groups obtained by flow cytometry were compared with the results from HPLC pigment fingerprinting. Both methods yield the percentage contribution of the different algal groups to total chl a. The chl a specific absorption coefficient of the phytoplankton (a à Phy ) was determined via visible (VIS) spectroscopy of samples taken from a hypertrophic lake (Auensee) in 2003. The results indicated that a à Phy of the total cell suspension is dependent on the phytoplankton structure as well as on environmental factors. The linear relationship between a à Phy at 675 nm and the product of the chl a content per cell and the biovolume offered the possibility to normalize phytoplankton absorption spectra to acquire the taxon-specific a à Phy . The estimated a à Phy (675 nm) values were used to normalize single cell absorption spectra at this wavelength to obtain the a à Phy between 400 and 750 nm for representatives of the major algal groups. Our measurements show that the absorption coefficient for the whole phytoplankton community varies within the season. Finally, we used the a à Phy and the chl a distribution to calculate the light absorption of each algal group in the hypertrophic lake.