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The ketocarotenoid fucoxanthin and its derivatives can absorb blue–green light enriched in marine environments. Fucoxanthin is widely adopted by phytoplankton species as a main light-harvesting pigment, in contrast to land plants that primarily employ chlorophylls. Despite its supreme abundance in the oceans, the last steps of fucoxanthin biosynthesis have remained elusive. Here, we identified the carotenoid isomerase-like protein CRTISO5 as the diatom fucoxanthin synthase that is related to the carotenoid cis–trans isomerase CRTISO from land plants but harbors unexpected enzymatic activity. A crtiso5 knockout mutant in the model diatom Phaeodactylum tricornutum completely lacked fucoxanthin and accumulated the acetylenic carotenoid phaneroxanthin. Recombinant CRTISO5 converted phaneroxanthin into fucoxanthin in vitro by hydrating its carbon–carbon triple bond, instead of functioning as an isomerase. Molecular docking and mutational analyses revealed residues essential for this activity. Furthermore, a photophysiological characterization of the crtiso5 mutant revealed a major structural and functional role of fucoxanthin in photosynthetic pigment–protein complexes of diatoms. As CRTISO5 hydrates an internal alkyne physiologically, the enzyme has unique potential for biocatalytic applications. The discovery of CRTISO5 illustrates how neofunctionalization leads to major diversification events in evolution of photosynthetic mechanisms and the prominent brown coloration of most marine photosynthetic eukaryotes.

Fucoxanthin is a major light-harvesting pigment in ecologically important algae such as diatoms, haptophytes, and brown algae (Phaeophyceae). Therefore, it is a major driver of global primary productivity. Species of these algal groups are brown colored because the high amounts of fucoxanthin bound to the proteins of their photosynthetic machineries enable efficient absorption of green light. While the structure of these fucoxanthin-chlorophyll proteins has recently been resolved, the biosynthetic pathway of fucoxanthin is still unknown. Here, we identified two enzymes central to this pathway by generating corresponding knockout mutants of the diatom Phaeodactylum tricornutum that are green due to the lack of fucoxanthin. Complementation of the mutants with the native genes or orthologs from haptophytes restored fucoxanthin biosynthesis. We propose a complete biosynthetic path to fucoxanthin in diatoms and haptophytes based on the carotenoid intermediates identified in the mutants and in vitro biochemical assays. It is substantially more complex than anticipated and reveals diadinoxanthin metabolism as the central regulatory hub connecting the photoprotective xanthophyll cycle and the formation of fucoxanthin. Moreover, our data show that the pathway evolved by repeated duplication and neofunctionalization of genes for the xanthophyll cycle enzymes violaxanthin de-epoxidase and zeaxanthin epoxidase. Brown algae lack diadinoxanthin and the genes described here and instead use an alternative pathway predicted to involve fewer enzymes. Our work represents a major step forward in elucidating the biosynthesis of fucoxanthin and understanding the evolution, biogenesis, and regulation of the photosynthetic machinery in algae.

Characterizing photosynthetic productivity is necessary to understand the ecological contributions and biotechnology potential of plants, algae, and cyanobacteria. Light capture efficiency and photophysiology have long been characterized by measurements of chlorophyll fluorescence dynamics. However, these investigations typically do not consider the metabolic network downstream of light harvesting. By contrast, genome-scale metabolic models capture species-specific metabolic capabilities but have yet to incorporate the rapid regulation of the light harvesting apparatus. Here, we combine chlorophyll fluorescence parameters defining photosynthetic and non-photosynthetic yield of absorbed light energy with a metabolic model of the pennate diatom Phaeodactylum tricornutum. This integration increases the model predictive accuracy regarding growth rate, intracellular oxygen production and consumption, and metabolic pathway usage. Through the quantification of excess electron transport, we uncover the sequential activation of non-radiative energy dissipation processes, cross-compartment electron shuttling, and non-photochemical quenching as the rapid photoacclimation strategy in P. tricornutum. Interestingly, the photon absorption thresholds that trigger the transition between these mechanisms were consistent at low and high incident photon fluxes. We use this understanding to explore engineering strategies for rerouting cellular resources and excess light energy towards bioproducts in silico. Overall, we present a methodology for incorporating a common, informative data type into computational models of light-driven metabolism and show its utilization within the design-build-test-learn cycle for engineering of photosynthetic organisms.

Experiments that longitudinally collect RNA sequencing (RNA-seq) data can provide transformative insights in biology research by revealing the dynamic patterns of genes. Such experiments create a great demand for new analytic approaches to identify differentially expressed (DE) genes based on large-scale time-course count data. Existing methods, however, are suboptimal with respect to power and may lack theoretical justification. Furthermore, most existing tests are designed to distinguish among conditions based on overall differential patterns across time, though in practice, a variety of composite hypotheses are of more scientific interest. Finally, some current methods may fail to control the false discovery rate. In this paper, we propose a new model and testing procedure to address the above issues simultaneously. Specifically, conditional on a latent Gaussian mixture with evolving means, we model the data by negative binomial distributions. Motivated by Storey (2007) and Hwang and Liu (2010), we introduce a general testing framework based on the proposed model and show that the proposed test enjoys the optimality property of maximum average power. The test allows not only identification of traditional DE genes but also testing of a variety of composite hypotheses of biological interest. We establish the identifiability of the proposed model, implement the proposed method via efficient algorithms, and demonstrate its good performance via simulation studies. The procedure reveals interesting biological insights, when applied to data from an experiment that examines the effect of varying light environments on the fundamental physiology of the marine diatom Phaeodactylum tricornutum.

Individual cells of cyanobacteria or algae are supplied with light in a highly irregular fashion when grown in industrial-scale photobioreactors (PBRs). These conditions coincide with significant reductions in growth rate compared to the static light environments commonly used in laboratory experiments. We grew a dense culture of the model cyanobacterium Synechocystis sp. PCC 6803 under a sinusoidal light regime in a bench-top PBR (the Phenometrics environmental PBR [ePBR]). We developed a computational fluid dynamics model of the ePBR, which predicted that individual cells experienced rapid fluctuations (∼6 s) between 2,000 and

This paper presents a case study on a 275 kV oil-filled cable. The condition assessment and diagnosis are based on analysis of cable surface temperature in relation to its current load and insulation dielectric loss. The work was initiated by a local abnormal temperature rise of 5.2 °C in cable surface temperature, which was observed during a routine inspection. The temperature rise occurred at bend area with a length of approximately one metre in the Blue phase. No PD activity was identified using on-line PD measurement. The relation between cable surface temperature, cable core temperature and cable insulation condition was then simulated based on the thermal model of power cables. According to simulation analysis, poor condition of cable insulation or oil from an oil duct penetrating a region under the cable surface were identified as possible reasons for the problem observed. An in service X-ray scanning technique was employed for further investigation and to aid diagnosis. The X-ray images revealed a slight distortion of the PVC sheath and the presence of multiple voids between cable insulation paper and the lead sheath. It was concluded that an oil leakage from the oil duct to the voids under the cable lead sheath was responsible for the local cable surface temperature rise. The result removed the concern of incipient cable breakdown, and a potential unplanned outage.

International audience; Photoacclimation consists of short-and long-term strategies used by photosynthetic organisms to adapt to dynamic light environments. Observable photophysiology changes resulting from these strategies have been used in coarse-grained models to predict light-dependent growth and photosynthetic rates. However, the contribution of the broader metabolic network , relevant to species-specific strategies and fitness, is not accounted for in these simple models. We incorporated photophysiology experimental data with genome-scale modeling to characterize organism-level, light-dependent metabolic changes in the model diatom Phaeodactylum tricornutum. Oxygen evolution and photon absorption rates were combined with condition-specific biomass compositions to predict metabolic pathway usage for cells acclimated to four different light intensities. Photorespiration, an ornithine-glutamine shunt, and branched-chain amino acid metabolism were hypothesized as the primary intercompartment reductant shuttles for mediating excess light energy dissipation. Additionally, simulations suggested that carbon shunted through photorespiration is recycled back to the chloroplast as pyruvate, a mechanism distinct from known strategies in photosynthetic organisms. Our results suggest a flexible metabolic network in P. tricornutum that tunes intercompart-ment metabolism to optimize energy transport between the organelles, consuming excess energy as needed. Characterization of these intercompartment reductant shuttles broadens our understanding of energy partitioning strategies in this clade of ecologically important primary producers.

This study proposes a novel integration of a municipal wastewater treatment facility (WWTF) with a cyanobacterial nutrient removal process for sidestream wastewater treatment. A life cycle assessment (LCA) approach was used to determine the effectiveness and environmental performances of the integrated system. The LCA is populated by models of wastewater process engineering, material balance, cyanobacterial growth, and kinetics of anaerobic digestion. The cyanobacteria growth model incorporates chlorophyll synthesis, nitrogen uptake, photosynthesis, centrate inhibition, and competition for nitrogen between cyanobacteria and nitrifiers. Modeling results are validated against experiments with Synechocystis sp. PCC6803 grown in sludge centrate. With a maximum specific growth rate of 1.09 day−1, the nitrogen removal rate of the proposed WWTF would be increased by 15% when compared to the baseline wastewater treatment facility with a biological nutrient removal process. Incorporating the cyanobacterial nutrient removal process as the sidestream wastewater treatment of a conventional activated sludge process reduces the total nitrogen concentrations discharged from the WWTF from 25.9 to 15.2 mg 1−1. Methane yield was found to be increased by 4% of the baseline value when cyanobacterial biomass was co-digested with the activated sludge. Life cycle energy use and greenhouse gas emissions were found to be reduced by 8% and 17%, respectively, relative to a baseline wastewater treatment facility. Overall, a cyanobacteria-based sidestream municipal wastewater treatment process could be an effective and environmentally sustainable biological nutrient removal process in the future addressing the water-energy-food nexus.

International audience; Photoautotrophic growth in nature requires the accumulation of energy-containing molecules via photosynthesis during daylight to fuel nighttime catabolism. Many diatoms store photosynthate as the neutral lipid triacylglycerol (TAG). While the pathways of diatom fatty acid and TAG synthesis appear to be well conserved with plants, the pathways of TAG catabolism and downstream fatty acid beta-oxidation have not been characterised in diatoms.We identified a putative mitochondria-targeted, bacterial-type acyl-CoA dehydrogenase (PtMACAD1) that is present in Stramenopile and Hacrobian eukaryotes, but not found in plants, animals or fungi. Gene knockout, protein-YFP tags and physiological assays were used to determine PtMACAD1's role in the diatom Phaeodactylum tricornutum.PtMACAD1 is located in the mitochondria. Absence of PtMACAD1 led to no consumption of TAG at night and slower growth in light : dark cycles compared with wild-type. Accumulation of transcripts encoding peroxisomal-based beta-oxidation did not change in response to day : night cycles or to PtMACAD1 knockout. Mutants also hyperaccumulated TAG after the amelioration of N limitation.We conclude that diatoms utilise mitochondrial beta-oxidation; this is in stark contrast to the peroxisomal-based pathways observed in plants and green algae. We infer that this pattern is caused by retention of catabolic pathways from the host during plastid secondary endosymbiosis.

Desmodesmus armatus is an emerging biofuel platform producing high amount of lipids and biomass in mass culture. We observed D. armatus in light-limiting, excess light and sinusoidal light environments to investigate its photoacclimation behaviors and the mechanisms by which it dissipates excess energy. Chlorophyll a:b ratios and the functional absorption cross section of photosystem II (PSII) suggested a constitutively small light harvesting antenna size relative to other green algae. In situ and ex situ measurements of photo-physiology revealed that nonphotochemical quenching (NPQ) is not a significant contributor to photoprotection, but cells do not suffer substantial photoinhibition despite its near absence. We performed membrane inlet mass spectrometry analysis to show that D. armatus has a very high capacity for alternative electron transport (AET) measured as light dependent oxygen consumption. Up to 90% of electrons generated at PSII can be dissipated by AET in a water-water cycle during growth in rapidly fluctuating light environments like those found in industrial scale photobioreactors. This work highlights the diversity of photoprotective mechanisms shown in algal systems, that NPQ is not necessarily required for effective photoprotection in some algae and suggests that engineering AET may be an attractive target for increasing biomass productivity of some strains.One-sentence summaryConstitutive small antennae, alternative electron transport and an efficient photosystem II turnover capacity enable D. armatus to photosynthesize efficiently.

A series of experiments were performed to evaluate the growth of the model cyanobacterium Synechocystis sp. PCC6803 cultured on wastewater centrate sourced from a municipal water reclamation facility. Centrate is the liquid removed from sludge during sewage processing. The addition of 304 μM Na2SO4 to centrate diluted by 90% in water yielded final cell concentrations that were over 6 times higher than cultures grown on just centrate. This suggested that S is the primary limiting nutrient for photoautorophic growth on centrate. The expression of the sulfate transporter system encoded by the spbA-cysTWA operon was upregulated when cells were grown in non-amended centrate, confirming that Synechocystis sp. PCC6803 experienced S-limitation during growth on centrate alone. Elemental analysis of centrate further confirmed that the proportion of S relative to other macronutrients is lower in centrate compared to the measured elemental ratios found in cyanobacterial biomass. The cyanobacteria removed 69% of the total soluble nitrogen (TN) in S-amended centrate compared to 25% from cultures grown in centrate alone. It is proposed that S could be recycled from the production of H2S during anaerobic digestion of wastewater, or it could be added from exogenous material such as gypsum. Overall, this work suggests that S-amendment of centrate could improve the sustainability of wastewater remediation and biomass production using photosynthetic microbes.

The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b6 f complex (Cytb6 f), which impacts linear and cyclic electron transport, but does not compromise the ability of the strain to perform state transitions. Furthermore, CPLD49 strongly associates with thylakoid membranes where it may be part of a membrane protein complex with another GreenCut protein, CPLD38; a mutant null for CPLD38 also impacts Cytb6 f complex accumulation. We investigated several potential functions of CPLD49, with some suggested by protein homology. Our findings are congruent with the hypothesis that CPLD38 and CPLD49 are part of a novel thylakoid membrane complex that primarily modulates accumulation, but also impacts the activity of the Cytb6 f complex. Based on motifs of CPLD49 and the activities of other CPLD49-like proteins, we suggest a role for this putative dehydrogenase in the synthesis of a lipophilic thylakoid membrane molecule or cofactor that influences the assembly and activity of Cytb6 f.

Measurements of cellular carbon assimilation and chlorophyll a fluorescence are key tools for assessing the physiological capacity of algae and cyanobacteria. The uptake of CO2 from the immediate environment informs a researcher about photosynthetic rates. Chlorophyll a fluorescence dynamics can be used to infer efficiencies about light energy harvesting efficiencies and loss processes such as the loss of energy as heat through non-photochemical quenching. Here we report the utilization of novel instrument that continuously measures CO2 exchange from an aqueous solution to suspended cells synchronously with chlorophyll fluorescence. We apply the instrument to investigate the response of photosynthesis to increasing light in 3 different productive strains of eukaryotic microalgae. We found that the carbon assimilation rates of Scenedesmus obliquus (UTEX393) did not saturate, even in light fluxes approximating full sunlight. Monoraphidium sp. (26BAM) had a high nonphotochemical quenching capacity in excess light. Both of these strains, and a third, Scendesmus actus (LRBOY07), demonstrated remarkably linear relationships between carbon assimilation rates and relative electron transport estimates at lower light intensities. This work shows the utility of novel instrumentation to quickly assay the photosynthetic capabilities of microalgal strains.

There is great interest in engineering photoautotrophic metabolism to generate bioproducts of societal importance. Despite the success in employing genome-scale modeling coupled with flux balance analysis to engineer heterotrophic metabolism, the lack of proper constraints necessary to generate biologically realistic predictions has hindered broad application of this methodology to phototrophic metabolism. Here we describe a methodology for constraining genome-scale models of photoautotrophy in the cyanobacteria Synechococcus elongatus PCC 7942. Experimental photophysiology parameters coupled to genome-scale flux balance analysis resulted in accurate predictions of growth rates and metabolic reaction fluxes at low and high light conditions. Additionally, by constraining photon uptake fluxes, we characterized the metabolic cost of excess excitation energy. The predicted energy fluxes were consistent with known light-adapted phenotypes in cyanobacteria. Finally, we leveraged the modeling framework to characterize existing photoautotrophic and photomixtotrophic engineering strategies for 2,3-butanediol production in S. elongatus. This methodology, applicable to genome-scale modeling of all phototrophic microorganisms, can facilitate the use of flux balance analysis in the engineering of light-driven metabolism.

Non-photochemical quenching, alternative electron transport and respiration all reduce photosynthetic efficiency. Targeted or random mutagenesis engineering approaches affecting genes involved in these processes have been repeatedly utilized to identify proteins which are essential for maximum photosynthetic efficiency. Ethylmethane sulfonate (EMS) was used here to randomly mutagenize wild type (WT) Desmodesmus armatus (SE00107). A chlorophyll a fluorescence-based screening procedure was utilized to analyse 6662 novel mutant strains and mutants with altered fluorescence trace kinetics, indicative of altered electron transport or photoprotection relative to WT. Mutants were subsequently analysed for improved biomass productivity. Cells were grown under a variety of experimental conditions, leading to the selection of mutants with increased photosynthetic parameters and growth rates relative to WT. Three of these mutants demonstrated on average 20, 28 and 37% improved biomass production compared to WT cultures in conditions that mimic outdoor cultivation. This research provides a framework for independent groups looking to rapidly isolate mutants with enhanced biomass productivity. It is also particularly relevant when working with organisms that have limited genomic sequencing available.

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.

Cable condition monitoring techniques play a key role in informing operators of the state of cable assets. Instead of using traditional methods such as partial discharge and dielectric loss measurements, this paper presents a method for cable condition assessment via the measurement and analysis of cable surface temperature. First, the theory underpinning the calculation of cable surface temperature is introduced. Then the cable surface temperature rise in relation to cable dielectric loss is presented. The case of a 275kV oil-filled cable where a local surface temperature rise of $5^{o}{C}$ was observed is also investigated in the paper. Results indicate two reasons possibly responsible for the temperature rise. Finally, the calculations of cable temperature in relation to insulation degradation for 11kV cables show that diagnosis of MV PILC and EPR cables relying on cable surface temperature rise is possible.

An in vitro urease enzyme assay was developed for the marine diatoms Thalassiosira pseudonana Hasle et Heimdal (clone 3H) and T. weissflogii (Grunow) Fryxell et Hasle (clone Actin). This assay involves the colorimetric measurement of ammonium following the hydrolysis of urea in crude cell homogenates and it is the first assay to account for the rate of nitrogen assimilation in both species grown on urea as the sole nitrogen source. Urease activity was found to be present regardless of nitrogen source, although activities showed distinctly different patterns depending on the species examined and form of nitrogen supplied. Under nitrogen-replete conditions, urease activity in T. pseudonana was present constitutively when grown on NH 4 1 and upregulated when grown on NO 3 2 or urea. In nitrogen-replete T. weissflogii , urease activity was present at high constitutive levels regardless of the nitrogen source and showed no upregulation. Nitrogen starvation did not upregulate activity in either species.

International audience; Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis (Arabidopsis thaliana) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome-b(6)f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency.

The GreenCut encompasses a suite of nucleus-encoded proteins with orthologs among green lineage organisms (plants, green algae), but that are absent or poorly conserved in non-photosynthetic/heterotrophic organisms. In Chlamydomonas reinhardtii, CPLD49 (Conserved in Plant Lineage and Diatoms49) is an uncharacterized GreenCut protein that is critical for maintaining normal photosynthetic function. We demonstrate that a cpld49 mutant has impaired photoautotrophic growth under high-light conditions. The mutant exhibits a nearly 90% reduction in the level of the cytochrome b

This paper presents a novel Partial Discharge (PD) localisation method, based on PD pulse Rise-Time and Transfer Function (RTTF), for use in on-line cable PD monitoring systems. This is based on the change in rise time as a function of cable length along which a PD pulse has propagated. A comparison with four other well-known location techniques, i.e. Time-Domain Reflectometry (TDR), Phase difference, Arrival Time Analysis (ATA) and Amplitude-Frequency (AF) mapping has been made and presented. Analysis shows that the TDR and Phase difference methods, although requiring a single measurement point, are only applicable to short length cable systems as both incident pulse and reflected pulse should be observable when it reaches the detection point. On the other hand, the ATA and AF mapping, which can be applied to longer cable systems, need to install detectors at multiple points and detect synchronously. In comparison, PD localisation by the proposed RTTF method, which requires detection of the incident pulse at only one measurement point, can be used to localise a PD source which is further away from the PD detection point than other methods and can be used in on-line PD monitoring systems. As on-line PD monitoring sensors commonly comprise of a high frequency current transformer (HFCT) and a high-pass filter, the rise time of detected PD pulses depends on the attenuation of the cable, the HFCT used and the filter applied. Simulations of pulse propagation in a cable and PD monitoring system are performed, based on analyses in the frequency domain using the concept of transfer functions. Results of RTTF from the simulations have been verified by laboratory experiments and on-site PD measurements, which provide evidence that the RTTF method is a very valuable and useful PD localisation technique.

The response of heterotrophic bacteria in a mesoscale iron (Fe) enrichment was measured in the northeast subarctic Pacific Ocean in July 2002. Addition of FeSO4 increased the dissolved Fe concentration in the fertilized patch to 2–3 nmol L−1 and triggered an increase in concentration of Fe(III)-binding ligands that complexed all of the dissolved Fe. Two to three days later, leucine incorporation rate and specific growth rate of bacteria doubled. Physiological markers of bacterial Fe nutritional state varied during the experiment as the microbial community assimilated the added Fe. Cellular uptake rate of an iron-siderophore complex, 55Fe-ferrioxamine B (FB), increased twofold to fourfold over background values and then declined by day 4.5. The fastest rate of Fe-FB uptake on day 2.5 coincided roughly with a transient increase in outer-membrane, 55Fe-FB-binding protein(s) in bacteria and with the peak in ligand concentration. Maximum potential uptake rate of inorganic Fe (Vmax) was 8 zmol Fe bacterium−1 h−1 prior to Fe enrichment and then decreased by a factor of four within 2.5 d of fertilizing the patch as bacteria became Fe sufficient. Vmax gradually increased by day 6.5 as the bacterial community re-entered iron deficiency. Similar changes in growth and Fe uptake kinetics were observed after a second Fe addition. Heterotrophic bacteria in the subarctic Pacific were Fe-deficient and responded directly to Fe addition by up-regulating pathways for Fe-siderophore acquisition and assimilating complexed Fe. The observation that increases in Fe uptake pathways and production were synchronous is consistent with the hypothesis that bacterial growth was directly limited by Fe.

Chlamydomonas reinhardtii insertion mutants disrupted for genes encoding acetate kinases (EC 2.7.2.1) (ACK1 and ACK2) and a phosphate acetyltransferase (EC 2.3.1.8) (PAT2, but not PAT1) were isolated to characterize fermentative acetate production. ACK1 and PAT2 were localized to chloroplasts, while ACK2 and PAT1 were shown to be in mitochondria. Characterization of the mutants showed that PAT2 and ACK1 activity in chloroplasts plays a dominant role (relative to ACK2 and PAT1 in mitochondria) in producing acetate under dark, anoxic conditions and, surprisingly, also suggested that Chlamydomonas has other pathways that generate acetate in the absence of ACK activity. We identified a number of proteins associated with alternative pathways for acetate production that are encoded on the Chlamydomonas genome. Furthermore, we observed that only modest alterations in the accumulation of fermentative products occurred in the ack1, ack2, and ack1 ack2 mutants, which contrasts with the substantial metabolite alterations described in strains devoid of other key fermentation enzymes.

Oxygenic photosynthesis is the process by which plants, algae, and cyanobacteria convert sunlight and CO 2 into chemical energy and biomass. Previously published estimates suggest that algal photosynthesis is, at best, able to convert approximately 5–7% of incident light energy to biomass and there is opportunity for improvement. Recent analyses of in situ photophysiology in mass cultures of algae and cyanobacteria show that cultivation methods can have detrimental effects on a cell's photophysiology – reinforcing the need to understand the complex responses of cell biology to a highly variable environment. A systems-based approach to understanding the stresses and efficiencies associated with light-energy harvesting, CO 2 fixation, and carbon partitioning will be necessary to make major headway toward improving photosynthetic yields.

Summary Diatoms are unicellular photosynthetic algae with promise for green production of fuels and other chemicals. Recent genome-editing techniques have greatly improved the potential of many eukaryotic genetic systems, including diatoms, to enable knowledge-based studies and bioengineering. Using a new technique, transcription activator-like effector nucleases (TALENs), the gene encoding the urease enzyme in the model diatom, Phaeodactylum tricornutum, was targeted for interruption. The knockout cassette was identified within the urease gene by PCR and Southern blot analyses of genomic DNA. The lack of urease protein was confirmed by Western blot analyses in mutant cell lines that were unable to grow on urea as the sole nitrogen source. Untargeted metabolomic analysis revealed a build-up of urea, arginine and ornithine in the urease knockout lines. All three intermediate metabolites are upstream of the urease reaction within the urea cycle, suggesting a disruption of the cycle despite urea production. Numerous high carbon metabolites were enriched in the mutant, implying a breakdown of cellular C and N repartitioning. The presented method improves the molecular toolkit for diatoms and clarifies the role of urease in the urea cycle.

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.

The LHCSR protein belongs to the light harvesting complex family of pigment-binding proteins found in oxygenic photoautotrophs. Previous studies have shown that this complex is required for the rapid induction and relaxation of excess light energy dissipation in a wide range of eukaryotic algae and moss. The ability of cells to rapidly regulate light harvesting between this dissipation state and one favoring photochemistry is believed to be important for reducing oxidative stress and maintaining high photosynthetic efficiency in a rapidly changing light environment. We found that a mutant of Chlamydomonas reinhardtii lacking LHCSR, npq4lhcsr1, displays minimal photoinhibition of photosystem II and minimal inhibition of short term oxygen evolution when grown in constant excess light compared to a wild type strain. We also investigated the impact of no LHCSR during growth in a sinusoidal light regime, which mimics daily changes in photosynthetically active radiation. The absence of LHCSR correlated with a slight reduction in the quantum efficiency of photosystem II and a stimulation of the maximal rates of photosynthesis compared to wild type. However, there was no reduction in carbon accumulation during the day. Another novel finding was that npq4lhcsr1 cultures underwent fewer divisions at night, reducing the overall growth rate compared to the wild type. Our results show that the rapid regulation of light harvesting mediated by LHCSR is required for high growth rates, but it is not required for efficient carbon accumulation during the day in a sinusoidal light environment. This finding has direct implications for engineering strategies directed at increasing photosynthetic productivity in mass cultures.

Partial discharge (PD) based condition monitoring has been widely applied to power cables. However, difficulties in interpretation of measurement results (location and criticality) remain to be tackled. This paper aims to develop further knowledge in PD signal propagation in power cables and attenuation by the PD monitoring system devices to address the localization and criticality issues. As on-line or in-service PD monitoring sensors commonly comprise of a high frequency current transformer (HFCT) and a high-pass filter, the characteristics of detected PD pulses depend on the attenuation of the cable, the HFCT used and the filter applied. Simulation of pulse propagation in a cable and PD monitoring system are performed, based on analyses in the frequency domain using the concept of transfer functions. Results have been verified by laboratory experiments and using on-site PD measurements. The knowledge gained from the research on the change in pulse characteristics propagating in a cable and through a PD detection system can be very useful to PD denoising and for development of a PD localization technique.

International audience; We have established a protocol that allows for parallel quantification of three carbohydrate pools in the marine diatom Phaeodactylum tricornutum. This method utilizes a series of extraction and digestion steps followed by the employment of the 3-methyl-2-benzothiazolinone hydrazone (MBTH) reducing sugar assay. Comparing carbohydrate content between hydrolyzed and non-hydrolyzed soluble extracts enables quantification of soluble, non-reducing carbohydrate. The latter fraction contains chrysolaminarin as verified by H-1 NMR and monomer composition of the purified glucan. We applied this method to investigate carbon partitioning in two experiments. We observed the accumulation of chrysolaminarin during the day and near complete consumption in the dark, supporting its role for fueling heterotrophic metabolism at night. We then observed little change in chrysolaminarin accumulation or consumption during nitrogen starvation, a condition that is known to increase the cellular content of the biofuel precursor triacylglycerol. Overall, this method improves the resolution of major carbohydrate pools in diatoms, complementing physiological and systems biology studies interested in exploring diatom metabolism.

© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Plants, algae and cyanobacteria need to regulate photosynthetic light harvesting in response to the constantly changing light environment. Rapid adjustments are required to maintain fitness because of a trade-off between efficient solar energy conversion and photoprotection. The xanthophyll cycle, in which the carotenoid pigment violaxanthin is reversibly converted into zeaxanthin, is ubiquitous among green algae and plants and is necessary for the regulation of light harvesting, protection from oxidative stress and adaptation to different light conditions 1,2. Violaxanthin de-epoxidase (VDE) is the key enzyme responsible for zeaxanthin synthesis from violaxanthin under excess light. Here we show that the Chlorophycean VDE (CVDE) gene from the model green alga Chlamydomonas reinhardtii encodes an atypical VDE. This protein is not homologous to the VDE found in plants and is instead related to a lycopene cyclase from photosynthetic bacteria 3. Unlike the plant-type VDE that is located in the thylakoid lumen, the Chlamydomonas CVDE protein is located on the stromal side of the thylakoid membrane. Phylogenetic analysis suggests that CVDE evolved from an ancient de-epoxidase that was present in the common ancestor of green algae and plants, providing evidence of unexpected diversity in photoprotection in the green lineage.

Liquid chromatography coupled to electrospray ionization-mass spectrometry (LC-ESI-MS) is a versatile and robust platform for metabolomic analysis. However, while ESI is a soft ionization technique, in-source phenomena including multimerization, nonproton cation adduction, and in-source fragmentation complicate interpretation of MS data. Here, we report chromatographic and mass spectrometric behavior of 904 authentic standards collected under conditions identical to a typical nontargeted profiling experiment. The data illustrate that the often high level of complexity in MS spectra is likely to result in misinterpretation during the annotation phase of the experiment and a large overestimation of the number of compounds detected. However, our analysis of this MS spectral library data indicates that in-source phenomena are not random but depend at least in part on chemical structure. These nonrandom patterns enabled predictions to be made as to which in-source signals are likely to be observed for a given compound. Using the authentic standard spectra as a training set, we modeled the in-source phenomena for all compounds in the Human Metabolome Database to generate a theoretical in-source spectrum and retention time library. A novel spectral similarity matching platform was developed to facilitate efficient spectral searching for nontargeted profiling applications. Taken together, this collection of experimental spectral data, predictive modeling, and informatic tools enables more efficient, reliable, and transparent metabolite annotation.

Light is the source of energy for oxygenic photosynthesis. Large variations in light availability expose photoautotrophic organisms to low fluxes that limit net photosynthesis and high fluxes that are in great excess of photosynthetic capacity. Plants, algae and cyanobacteria need to adjust their photosynthetic apparatus over the time scales of light variability - from seconds to seasons - to balance the energy needs of the organism and to avoid the photooxidative damage associated with excess light energy absorption (photoprotection). This chapter reviews recent discoveries regarding the light acclimation and photoprotective processes within the chloroplast and cyanobacteria. We focus on energy dissipation pathways and the signaling pathways associated with the response to excess light stress.

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.

To investigate the impact of iron deficiency on bioenergetic pathways in Chlamydomonas, we compared growth rates, iron content, and photosynthetic parameters systematically in acetate versus CO2-grown cells. Acetate-grown cells have, predictably (2-fold) greater abundance of respiration components but also, counter-intuitively, more chlorophyll on a per cell basis. We found that phototrophic cells are less impacted by iron deficiency and this correlates with their higher iron content on a per cell basis, suggesting a greater capacity/ability for iron assimilation in this metabolic state. Phototrophic cells maintain both photosynthetic and respiratory function and their associated Fe-containing proteins in conditions where heterotrophic cells lose photosynthetic capacity and have reduced oxygen evolution activity. Maintenance of NPQ capacity might contribute to protection of the photosynthetic apparatus in iron-limited phototrophic cells. Acetate-grown iron-limited cells maintain high growth rates by suppressing photosynthesis but increasing instead respiration. These cells are also able to maintain a reduced plastoquinone pool. Electronic supplementary material The online version of this article (doi:10.1007/s11120-010-9562-8) contains supplementary material, which is available to authorized users.

Light is necessary for photosynthesis, but its absorption by pigment molecules such as chlorophyll can cause severe oxidative damage and result in cell death. The excess absorption of light energy by photosynthetic pigments has led to the evolution of protective mechanisms that operate on the timescale of seconds to minutes and involve feedback-regulated de-excitation of chlorophyll molecules in photosystem II (qE). Despite the significant contribution of eukaryotic algae to global primary production, little is known about their qE mechanism, in contrast to that in flowering plants. Here we show that a qE-deficient mutant of the unicellular green alga Chlamydomonas reinhardtii, npq4, lacks two of the three genes encoding LHCSR (formerly called LI818). This protein is an ancient member of the light-harvesting complex superfamily, and orthologues are found throughout photosynthetic eukaryote taxa, except in red algae and vascular plants. The qE capacity of Chlamydomonas is dependent on environmental conditions and is inducible by growth under high light conditions. We show that the fitness of the npq4 mutant in a shifting light environment is reduced compared to wild-type cells, demonstrating that LHCSR is required for survival in a dynamic light environment. Thus, these data indicate that plants and algae use different proteins to dissipate harmful excess light energy and protect the photosynthetic apparatus from damage.

In higher plants, regulation of excess absorbed light is essential for their survival and fitness, as it enables avoidance of a build up of singlet oxygen and other reactive oxygen species. Regulation processes (known as non-photochemical quenching; NPQ) can be monitored by steady-state fluorescence on intact plant leaves. Pulse amplitude modulated (PAM) measurements of chlorophyll a fluorescence have been used for over 20 years to evaluate the amount of NPQ and photochemistry (PC). Recently, a quantum yield representation of NPQ ( Φ NPQ ), which incorporates a variable fraction of open reaction centers, was proposed by Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73]. In this work we extend the quantum yield approach to describe the yields of reversible energy-dependent quenching ( Φ qE ), state transitions to balance PC between photosystems II and I ( Φ qT ), and photoinhibition quenching associated with damaged reaction centers ( Φ qI ). We showed the additivity of the various quantum yield components of NPQ through experiments on wild-type and npq1 strains of Arabidopsis thaliana. The quantum yield approach enables comparison of Φ qE with data from a variety of techniques used to investigate the mechanism of qE. We showed that Φ qE for a series of A. thaliana genotypes scales linearly with the magnitude of zeaxanthin cation formation, suggesting that charge-transfer quenching is largely responsible for qE in plants.

Plastocyanin is an essential and abundant copper protein required for photosynthesis in higher plants. Severe copper deprivation has the potential to cause a defect in photosynthetic electron transport due to a lack in plastocyanin. The Cu-microRNAs, which are up-regulated under Cu deficiency, down-regulate the expression of target Cu proteins other than plastocyanin, cytochrome-c oxidase and the ethylene receptors. It has been proposed that this mechanism saves Cu for plastocyanin maturation. We aimed to test how hybrid poplar, a species that has capacity to rapidly expand its photosynthetically active tissue, responds to variations in Cu availability over time. Measurement of chlorophyll fluorescence after Cu depletion revealed a drastic effect on photosynthesis in hybrid poplar. The decrease in photosynthetic capacity was correlated with a reduction in plastocyanin protein levels. Compared to older leaves, plastocyanin decreased more strongly in developing leaves, which also lost more photosynthetic electron transport capacity. The effect of Cu depletion on older and more developed leaves was minor and these leaves maintained much of their photosynthetic capacity. Interestingly, upon resupply of Cu to the medium a very rapid recovery of Cu levels was seen in the younger leaves with a concomitant rise in the expression and activity of plastocyanin. In contrast, the expression of those Cu proteins, which are targets of microRNAs was under the same circumstances delayed. At the same time, Cu resupply had only minor effects on the older leaves. The data suggest a model where rapid recovery of photosynthetic capacity in younger leaves is made possible by a preferred allocation of Cu to plastocyanin in younger leaves, which is supported by Cu-microRNA expression.

Diatoms are responsible for 40% of ocean production and are strongly limited by the lack of iron salts in the sea. The shortage of this important nutrient may explain a surprising discovery: the oceanic diatom Thalassiosira oceanica uses a copper-containing plastocyanin for electron transport. All other chlorophyll c-containing taxa, including coastal diatoms, use the iron-containing cytochrome c6 instead. The use of these metalloproteins matches the availabilities of copper and iron in the ocean, and this new discovery suggests that copper is a potentially important nutrient in the open sea. The supply of some essential metals to pelagic ecosystems is less than the demand, so many phytoplankton have slow rates of photosynthetic production and restricted growth1. The types and amounts of metals required by phytoplankton depends on their evolutionary history2 and on their adaptations to metal availability3,4, which varies widely among ocean habitats. Diatoms, for example, need considerably less iron (Fe) to grow than chlorophyll-b-containing taxa2, and the oceanic species demand roughly one-tenth the amount of coastal strains5,6,7. Like Fe, copper (Cu) is scarce in the open sea, but notably higher concentrations of it are required for the growth of oceanic than of coastal isolates8. Here we report that the greater Cu requirement in an oceanic diatom, Thalassiosira oceanica, is entirely due to a single Cu-containing protein, plastocyanin, which—until now—was only known to exist in organisms with chlorophyll b and cyanobacteria. Algae containing chlorophyll c, including the closely related coastal species T. weissflogii, are thought to lack plastocyanin and contain a functionally equivalent Fe-containing homologue, cytochrome c6 (ref. 9). Copper deficiency in T. oceanica inhibits electron transport regardless of Fe status, implying a constitutive role for plastocyanin in the light reactions of photosynthesis in this species. The results suggest that selection pressure imposed by Fe limitation has resulted in the use of a Cu protein for photosynthesis in an oceanic diatom. This biochemical switch reduces the need for Fe and increases the requirement for Cu, which is relatively more abundant in the open sea.

Centric diatoms isolated from open ocean environments require higher concentrations of Cu for growth than their coastal counterparts. In artificial seawater medium containing ,1 nmol L 21 Cu, three coastal species maintained near maximum rates of growth, but the oceanic clones were unable to survive. Copper limitation was more severe in the diatoms grown in low- than in high-Fe seawater, suggesting that Cu and Fe were interacting essential resources. The interactive effect was in part the result of a Cu requirement for Fe transport. Thalassiosira weissflogii and Thalassiosira oceanica had lower Fe quotas and slower rates of Fe uptake when [Cu] was reduced in the medium. Brief exposure of Cu-limited cells to 10 nmol L 21 Cu increased the instantaneous Fe uptake rate by 1.5 times in T. oceanica. Steady-state uptake rates of both species at high, growth-saturating concentrations of Fe were also Cu dependent. Oceanic species appeared to have an additional Cu requirement that was independent of Fe acquisition and likely responsible for their higher requirements compared to coastal species. Evidence for the importance of Cu in natural communities of phytoplankton was obtained from an incubation experiment performed in the Fe-limited basin of the Bering Sea. Addition of 2 nmol L 21 Cu doubled the phytoplankton net growth rate compared to the untreated controls and, in the presence of extra Fe, increased the growth rate compared to the samples amended with Fe alone. The results suggest that Cu may be an important micronutrient for phytoplankton growth in low-Fe regions of the sea because of its role in Fe acquisition. Paradoxically, oceanic diatoms may be more susceptible to the effects of low Cu concentrations than coastal species.

We have discovered that coastal and oceanic diatoms require more manganese (Mn) to grow in iron (Fe) -deficient than in Fe-sufficient seawater. At low inorganic concentrations, like those of the open sea, Fe and Mn can thus colimit Thalassiosira pseudonana and T. oceanica so that maximum rates of cell division are achieved only when both resources are added simultaneously to cultures. Colimited diatoms amended with either Fe or Mn alone show unique physiological responses, which implies that the observed interaction between Fe and Mn is not caused by a substitution of one metal for the other. Iron deficiency increases the Mn quota of T. pseudonana by three times compared with controls and enhances the production of reactive oxygen species by 1.7 times in T. weissflogii. Both diatoms respond to this oxidative stress by increasing the activities of the antioxidant enzyme superoxide dismutase (SOD). The Mn content of the SODs increases by 1.8 to 2.8 times when Fe is limiting, which suggests that the SODs contain Mn and may account for part of the observed increase in the Mn quota. Such an increased biochemical requirement may elevate the Mn content of low Fe diatoms, and possibly other phytoplankton, resulting in high Mn: Fe ratios in surficial particulate matter in Fe-limited regions of the sea.

Absorption of light in excess of the capacity for photosynthetic electron transport is damaging to photosynthetic organisms. Several mechanisms exist to avoid photodamage, which are collectively referred to as nonphotochemical quenching. This term comprises at least two major processes. State transitions (qT) represent changes in the relative antenna sizes of photosystems II and I. High energy quenching (qE) is the increased thermal dissipation of light energy triggered by lumen acidification. To investigate the respective roles of qE and qT in photoprotection, a mutant (npq4 stt7-9) was generated in Chlamydomonas reinhardtii by crossing the state transition–deficient mutant (stt7-9) with a strain having a largely reduced qE capacity (npq4). The comparative phenotypic analysis of the wild type, single mutants, and double mutants reveals that both state transitions and qE are induced by high light. Moreover, the double mutant exhibits an increased photosensitivity with respect to the single mutants and the wild type. Therefore, we suggest that besides qE, state transitions also play a photoprotective role during high light acclimation of the cells, most likely by decreasing hydrogen peroxide production. These results are discussed in terms of the relative photoprotective benefit related to thermal dissipation of excess light and/or to the physical displacement of antennas from photosystem II.

New insights into algal biology have recently emerged from the sequencing of the genomes of three unicellular green algae. Although green algae and plants share many of the same core metabolic pathways, analysis of the Chlamydomonas reinhardtii genome suggests retention of many genes from its