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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.

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.

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 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.

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.

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.