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International audience; Photosynthetic microbes respond to changing light environments to balance photosynthetic process with light induced damage and photoinhibition. There have been very few characterizations of photosynthetic physiology or biomass partitioning during the day in mass culture. Understanding the constraints on photosynthetic efficiency and biomass accumulation are necessary for engineering superior strains or cultivation methods. We observed the photosynthetic physiology of nutrient replete Phaeodactylum tricornutum growing in light environments that mimic those found in rapidly mixing, outdoor, low biomass photobioreactors. We found little evidence for photoinhibition or non-photochemical quenching in situ, suggesting photosynthesis remains highly efficient throughout the day. Cells doubled their organic carbon from dawn to dusk and a small percentage-around 20%-of this carbon was allocated to carbohydrates or triacylglycerol. We conclude that the self-shading provided by dense culturing of P. tricornutum inhibits the induction of photodamage, and energy dissipation processes that would otherwise lower productivity in an outdoor photobioreactor.

Phaeodactylum tricornutum is a widely used model organism for studying diatom biology. We created a P. tricornutum-specific literature database, or bibliome, that can be interactively annotated by users to improve the relevance and accuracy of search queries. A bibliome represents the current knowledge about a specific organism or biological process and is the foundation for genome-scale computation models. The bibliome of P. tricornutum was assembled from literature searches of ‘P. tricornutum’ (as topic) in two different existing databases, the Web of Knowledge and PubMed resulting in 2497 independent articles. We manually curated the bibliome for relevance to biochemical, physiological, and molecular biology studies. Literature that described the use of P. tricornutum as feed source and ecotoxicology studies were excluded from the bibliome. This process resulted in a final list of 1124 entries – 20% of which received additional classification in the form of notes or keywords. We present the bibliome in an HTML format that is easily searched and that can be customized according to the interests of individual researchers. This HTML tool can also easily be adapted for other algal species or specific research topics.

Diatoms are eukaryotic microalgae that contain genes from various sources, including bacteria and the secondary endosymbiotic host. Due to this unique combination of genes, diatoms are taxonomically and functionally distinct from other algae and vascular plants and confer novel metabolic capabilities. Based on the genome annotation, we performed a genome-scale metabolic network reconstruction for the marine diatom Phaeodactylum tricornutum. Due to their endosymbiotic origin, diatoms possess a complex chloroplast structure which complicates the prediction of subcellular protein localization. Based on previous work we implemented a pipeline that exploits a series of bioinformatics tools to predict protein localization. The manually curated reconstructed metabolic network iLB1027_lipid accounts for 1,027 genes associated with 4,456 reactions and 2,172 metabolites distributed across six compartments. To constrain the genome-scale model, we determined the organism specific biomass composition in terms of lipids, carbohydrates, and proteins using Fourier transform infrared spectrometry. Our simulations indicate the presence of a yet unknown glutamine-ornithine shunt that could be used to transfer reducing equivalents generated by photosynthesis to the mitochondria. The model reflects the known biochemical composition of P. tricornutum in defined culture conditions and enables metabolic engineering strategies to improve the use of P. tricornutum for biotechnological applications.

The effects of 4E/Z-octadienal (OCTA) on ScDSP-1 and ScDSP-2 gene expression and reactive oxygen species (ROS) production were investigated in the marine diatom Skeletonema tropicum (formerly costatum) using qRTPCR and flow cytometry. ScDSP-1 and ScDSP-2 genes have been previously shown to be involved in cell death in ageing cells and in response to photosynthetic stress. OCTA induced a differential, concentration-dependent DSP gene expression associated to ROS production, 821.6 and 97.7 folds higher for ScDSP-1 and ScDSP-2, respectively. Among the concentrations tested, only 8 μM OCTA, which caused a reduction of 50% in cell concentrations at 24 h, was able to elicit an expression pattern consistent with a signalling role. Interestingly, only intermediate levels of reactive oxygen species (ROS) (i.e., 1.5±0.1 increase) were observed to be elicited by such concentration. These results suggest that ROS are key components of the molecular cascade triggered by polyunsaturated aldehydes (PUA) and leading to cell death. This could have implications for bloom final stages at sea, where PUA may act as effectors of diatom population dynamics through ROS acting as modulators.

Polyunsaturated aldehydes (PUA) have recently been shown to induce reactive oxygen species (ROS) and possibly reactive nitrogen species (RNS, e.g., peroxynitrite) in the diatom Skeletonema marinoi (S. marinoi), which produces high amounts of PUA. We now are attempting to acquire better understanding of which reactive molecular species are involved in the oxidative response of S. marinoi to PUA. We used flow cytometry, the dye dihydrorhodamine 123 (DHR) as the main indicator of ROS (but which is also known to partially detect RNS), and different scavengers and inhibitors of both nitric oxide (NO) synthesis and superoxide dismutase activity (SOD). Both the scavengers Tempol (for ROS) and uric acid (UA, for peroxynitrite) induced a lower DHR-derived green fluorescence in S. marinoi cells exposed to the PUA, suggesting that both reactive species were produced. When PUA-exposed S. marinoi cells were treated with the NO scavenger 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), an opposite response was observed, with an increase in DHR-derived green fluorescence. A higher DHR-derived green fluorescence was also observed in the presence of sodium tungstate (ST), an inhibitor of NO production via nitrate reductase. In addition, two different SOD inhibitors, 2-methoxyestradiol (2ME) and sodium diethyldithiocarbamate trihydrate (DETC), had an effect, with DETC inducing the strongest inhibition after 20 min. These results indicate the involvement of O2 (•) generation and SOD activity in H2 O2 formation (with downstream ROS generation dependent from H2 O2 ) in response to PUA exposure. This is relevant as it refines the biological impact of PUA and identifies the specific molecules involved in the response. It is speculated that in PUA-exposed S. marinoi cells, beyond a certain threshold of PUA, the intracellular antioxidant system is no longer able to cope with the excess of ROS, thus resulting in the observed accumulation of both O2 (•-) and H2 O2 . This might be particularly relevant for population dynamics at sea, during blooms, when cell lysis increases and PUA are released. It can be envisioned that in the final stages of blooms, higher local PUA concentrations accumulate, which in turn induces intracellular ROS generation that ultimately leads to cell death and bloom decay.

Two oceanographic cruises encompassing the whole Adriatic Sea were carried out during February and October 2008. Selected stations were sampled at several depths to determine total prokaryotes and picocyanobacteria abundance using epifluorescence microscopy, and to estimate prokaryotic carbon production by 3 H-leucine incorporation. Biological data were related to physical parameters including temperature, salinity and fluorescence, and an attempt to associate bacterial dynamics to water mass characteristics was performed. In both seasons prokaryotic distribution and production showed a decreasing latitudinal gradient likely dependent on riverine inputs highlighted by a strong negative correlation with salinity ( P

Marine photoheterotrophic microorganisms are capable of using light to meet their energy requirements and organic compounds as both carbon and energy sources. We still have little knowledge of the extent to which stimulation of these microorganisms by light could affect the estimates of organic carbon uptake. We evaluated the light and dark prokaryotic organic carbon uptake (H-3-leucine) rates in a grid of stations covering the whole Adriatic Sea during winter and late summer. Light-exposed (photosynthetically active radiation and ultraviolet radiation [UVR]) surface samples were either unaffected, photostimulated or photoinhibited without any clear geographical or seasonal pattern. Light-enhanced leucine uptake occurred only in 23% of assays, suggesting that photoheterotrophy is not a major metabolic strategy and/or it is often counterbalanced by negative effects caused by UVR. Concentrations of bacteriochlorophyll a, zeaxanthin and divinyl chlorophyll a were measured in order to relate 3H-leucine uptake to the distribution of aerobic anoxygenic photo trophs (AAPs), total Cyanobacteria and Prochlorococcus populations, respectively, together with direct estimates of total prokaryotes and Synechococcus abundance. No relationship between light-enhanced leucine uptake and presence of picocyanobacteria or AAPs was evident. Divinyl chlorophyll a concentration was below the limit of detection in February, whereas in late summer the highest values were found around 50 m depth in the central and southern basins. In contrast, bacteriochlorophyll a concentration was correlated to total prokaryote abundance and dissolved organic carbon. Since locally leucine uptake in the light was markedly different from dark controls, in situ light incubations should be more appropriate than dark incubations when carbon budget calculations are intended.

Nitric oxide (NO) and reactive oxygen species (ROS) production was investigated in the marine diatom, Skeletonema marinoi (SM), exposed to 2E,4E/Z-decadienal (DECA), 2E,4E/Z-octadienal (OCTA), 2E,4E/Z-heptadienal (HEPTA) and a mix of these last two (MIX). When exposed to polyunsaturated aldehydes (PUA), a decrease of NO was observed, proportional to the PUA concentration (85% of the initial level after 180 min with 66 µM DECA). Only OCTA, HEPTA and MIX induced a parallel increase of ROS, the highest (2.9-times the control) with OCTA concentrations twice the EC50 for growth at 24 h (20 μM). The synthesis of carotenoids belonging to the xanthophyll cycle (XC) was enhanced during exposure, suggesting their antioxidant activity. Our data provide evidence that specific pathways exist as a reaction to PUA and that they depend upon the PUA used and/or the diatom species. In fact, Phaeodactylum tricornutum (PT) produces NO in response to DECA, but not to OCTA. We advance the hypothesis that SM perceives OCTA and HEPTA as intra-population infochemicals (as it produces PUA), while PT (non-PUA producing species) perceives them as allelochemicals. The ability to produce and to use PUA as infochemicals may underlie ecological traits of different diatom species and modulate ecological success in natural communities.