Getting the most out of it: optimal experiments for parameter estimation of microalgae growth models

International audienceMathematical models are expected to play a pivotal role for driving microalgal production towards a profitable process of renewable energy generation. To render models of microalgae growth useful tools for prediction and process optimization, reliable parameters need to be provided. This reliability implies a careful design of experiments that can be exploited for parameter estimation. In this paper, we provide guidelines for the design of experiments with high informative content based on optimal experiment techniques to attain an accurate parameter estimation. We study a real experimental device devoted to evaluate the effect of temperature and light on microalgae growth. On the basis of a mathematical model of the experimental system, the optimal experiment design problem was formulated and solved with both static (constant light and temperature) and dynamic (time varying light and temperature) approaches. Simulation results indicated that the optimal experiment design allows for a more accurate parameter estimation than that provided by the existing experimental protocol. For its efficacy in terms of the maximum likelihood properties and its practical aspects of implementation, the dynamic approach is recommended over the static approach

Modeling continuous cultures of microalgae colimited by nitrogen and phosphorus

International audienceIt is well documented that the combination of low nitrogen and phosphorus resources can lead to situations where colimitation of phytoplankton growth arises, yet the underlying mechanisms are not fully understood. Here, we propose a Droop-based model built on the idea that colimitation by nitrogen and phosphorus arises from the uptake of nitrogen. Indeed, since N-porters are active systems, they require energy that could be related to the phosphorus status of the cell. Therefore, we assumed that N uptake is enhanced by the P quota. Our model also accounts for the biological observations that uptake of a nutrient can be down-regulated by its own internal quota, and succeeds in describing the strong contrast for the non-limiting quotas under N-limited and P-limited conditions that was observed on continuous cultures with and with . Our analysis suggests that, regarding the colimitation concept, N and P would be better considered as biochemically dependent rather than biochemically independent nutrients

Deuterium in marine organic biomarkers: toward a new tool for quantifying aquatic mixotrophy

The traditional separation between primary producers (autotrophs) and consumers (heterotrophs) at the base of the marine food web is being increasingly replaced by the paradigm that mixoplankton, planktonic protists with the nutritional ability to use both phago (hetero)trophy and photo(auto)trophy to access energy are widespread globally. Thus, many ‘phytoplankton’ eat, while 50% of ‘protozooplankton’ also perform photosynthesis. Mixotrophy may enhance primary production, biomass transfer to higher trophic levels and the efficiency of the biological pump to sequester atmospheric CO2 into the deep ocean. Although this view is gaining traction, science lacks a tool to quantify the relative contributions of autotrophy and heterotrophy in planktonic protists. This hinders our understanding of their impacts on carbon cycling within marine pelagic ecosystems. It has been shown that the hydrogen (H) isotopic signature of lipids is uniquely sensitive to heterotrophy relative to autotrophy in plants and bacteria. Here, we explored whether it is also sensitive to the trophic status in protists. The new understanding of H isotope signature of lipid biomarkers suggests it offers great potential as a novel tool for quantifying the prevalence of mixotrophy in diverse marine microorganisms and thus for investigating the implications of the ‘mixoplankton’ paradigm

Lipids and lipolytic enzymes of the microalga Isochrysis galbana

International audienceMarine microalgae are now well-known for their ability to produce omega-3 long chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Among these microalgae, Isochrysis galbana has received increasing interest especially because of its high DHA content and its common use in hatchery to feed fish larvae and clams. Moreover, lipolysis occurring from the biomass harvest stage suggests that I. galbana may contain lipolytic enzymes with potential interesting selectivities. For these reasons, the potential of this microalga for the production of valuable lipids and lipolytic enzymes was investigated. Lipid analysis revealed that DHA is mainly located at the sn-2 position of the phospholipids. Thus, I. galbana was considered as an interesting starting material for the lipase catalyzed production of 1-lyso-2-DHA-phospholipids which are considered as convenient vehicles for the conveyance of DHA to the brain. Lipids from I. galbana can also be used for the enzyme-catalyzed production of structured phospholipids containing one DHA and one medium chain fatty acid in order to combine interesting therapeutic and biological benefits. Starting from total RNA extract from I. galbana, coding sequences of putative lipolytic enzymes were obtained by RACE and Nested PCR. The heterologous expression of a sequence designated IgTeCe was implemented. An expression plasmid was constructed by ligating the coding sequence to a plasmid vector and then cloned and expressed in E. coli. Results showed the effective functionality of plasmid construction for the production of a recombinant protein with the expected molecular mass. Moreover, local alignment using BLASTP and biochemical evidences support the hypothesis that the expressed protein is a thioesterase. Keywords: microalgae / v-3 polyunsaturated fatty acids / phospholipids / lipolytic enzymes Résumé-Lipides et enzymes lipolytiques de la microalgue Isochrysis galbana. Les microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série v-3 comme l'acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérêt croissant du fait d'un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D'autre part, la lipolyse souvent observée dès l'étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu'I. galbana pourrait contenir des enzymes lipolytiques intéressantes en termes de sélectivité. Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d'intérêt et d'enzymes lipolytiques a été étudié. L'analyse des lipides d'I. galbana a tout d'abord révélé que le DHA était majoritairement greffé sur la position sn-2 des phospholipides. Dans ce contexte, les phospholipides d'I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2-DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. À partir des lipides d'I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants. À partir des ARN totaux d'I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L'expression hétérologue d'une séquence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que laLes microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série ω-3 comme l’acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérèt croissant du fait d’un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D’autre part, la lipolyse souvent observée dès l’étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu’I. galbana pourrait contenir des enzymes lipolytiques intéressantes en terme de sélectivité.Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d’intérêt et d’enzymes lipolytiques a été étudié.L’analyse des lipides d’I. galbana a tout d’abord revélé que le DHA était majoritairement greffé sur la position sn-2 des phopholipides. Dans ce contexte, les phospholipids d’I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2–DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. A partir des lipides d’I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants.A partir des ARN totaux d’I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L’expression hétérologue d’une sequence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que la construction plasmidique permettait bien d’obtenir une protéine recombinante avec la masse moléculaire attendue. D’autre part, l’outil d’alignement local de séquences, BLASTP, ainsi que des données biochimiques ont permis de confirmer l’hypothèse que la protéine obtenue était une thioestérase

Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation

Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cyto-skeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity

Mixed microalgae culture for ammonium removal in the absence of phosphorus: Effect of phosphorus supplementation and process modeling

Microalgal growth and ammonium removal in a P-free medium have been studied in two batch photobioreactors seeded with a mixed microalgal culture and operated for 46 days. A significant amount of ammonium (106 mg NH4-Nl(-1)) was removed in a P-free medium, showing that microalgal growth and phosphorus uptake are independent processes. The ammonium removal rate decreased during the experiment, partly due to a decrease in the cellular phosphorus content. After a single phosphate addition in the medium of one of the reactors, intracellular phosphorus content of the corresponding microalgal culture rapidly increased, and so did the ammonium removal rate. These results show how the amount of phosphorus internally stored affects the ammonium removal rate. A mathematical model was proposed to reproduce these observations. The kinetic expression for microalgae growth includes a Monad term and a Hill's function to represent the effect of ammonium and stored polyphosphate concentrations, respectively. The proposed model accurately reproduced the experimental data (r=0.952, P-value <0.01).This research work has been supported by the Spanish Ministry of Economy and Competitiveness (MINECO, Projects CTM2011-28595-C02-01/02 jointly with the European Regional Development Fund (ERDF). They are both gratefully acknowledged.Ruiz Martinez, A.; Serralta Sevilla, J.; Paches Giner, MAV.; Seco Torrecillas, A.; Ferrer, J. (2014). Mixed microalgae culture for ammonium removal in the absence of phosphorus: Effect of phosphorus supplementation and process modeling. Process Biochemistry. 49(12):2249-2257. doi:10.1016/j.procbio.2014.09.002S22492257491

Testing the Waste Based Biorefinery Concept: Pilot Scale Cultivation of Microalgal Species on Spent Anaerobic Digestate Fluids

PurposeA waste based biorefinery approach has been tested.MethodsThis has been investigated by culturing in a 800 L photobioreactor two autotrophic microalgae namely Nannochloropsis oceanica and Scenedesmus quadricauda utilising filtered spent anaerobic digestate fluids of N:P ratio 14.22 as substrate.ResultsSignificant rates of bioremediation simultaneously with biomass and associated end product formation were achieved. Nitrogen and phosphorus of waste based media was decreased up to 90%. The biomass biochemical analysis of the microalgae when grown on the waste based formulated media demonstrated the comparable content of lipids and proteins with the species grown on f/2 media.ConclusionsTheoretical biomethane potential generation, should the algal cultures be placed in an anaerobic digester, was calculated at 0.58 L CH4 g−1 VS for N. oceanica and 0.48 L CH4 g−1 VS for S. quadricauda showing comparable results with other studies of different source of biomass

Continuous cultivation of photosynthetic microorganisms: approaches, applications and future trends

The possibility of using photosynthetic microorganisms, such as cyanobacteria and microalgae, for converting light and carbon dioxide into valuable biochemical products has raised the need for new cost-efficient processes ensuring a constant product quality. Food, feed, biofuels, cosmetics and pharmaceutics are among the sectors that can profit from the application of photosynthetic microorganisms. Biomass growth in a photobioreactor is a complex process influenced by multiple parameters, such as photosynthetic light capture and attenuation, nutrient uptake, photobioreactor hydrodynamics and gas-liquid mass transfer. In order to optimize productivity while keeping a standard product quality, a permanent control of the main cultivation parameters is necessary, where the continuous cultivation has shown to be the best option. However it is of utmost importance to recognize the singularity of continuous cultivation of cyanobacteria and microalgae due to their dependence on light availability and intensity. In this sense, this review provides comprehensive information on recent breakthroughs and possible future trends regarding technological and process improvements in continuous cultivation systems of microalgae and cyanobacteria, that will directly affect cost-effectiveness and product quality standardization. An overview of the various applications, techniques and equipment (with special emphasis on photobioreactors) in continuous cultivation of microalgae and cyanobacteria are presented. Additionally, mathematical modelling, feasibility, economics as well as the applicability of continuous cultivation into large-scale operation, are discussed.This research work was supported by the grant SFRH/BPD/98694/2013 (Bruno Fernandes) from Fundacao para a Ciencia e a Tecnologia (Portugal). The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013. The authors also thank the Project "BioInd Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" Co-funded by the Programa Operacional Regional do Norte (ON.2-O Novo Norte), QREN, FEDE

Rôle de ROR2 et de la Polarité Cellulaire Planaire dans la réponse des cellules endothéliales au flux sanguin

Endothelial cells (ECs) lining vessels sense and transduce mechanical forces created by blood flow into intracellular signals, and are especially sensitive to shear stress. Depending on the vascular area, ECs are exposed to different flow patterns: mainly laminar in straight part of arteries, and disturbed, considered as atheroprone, in regions of curvature or bifurcation. The objective of this thesis is to study the involvement of ROR2, a tyrosine kinase receptor known to regulate Planar Cell Polarity pathway, in endothelial responses to blood flow-induced shear stress.First, we focused on EC responses to laminar flow which induces cell elongation and polarization toward the flow axis and an atheroprotective phenotype. By in vivo approaches, using transgenic mice deleted for Ror2 specifically in ECs (Ror2iECKO), and in vitro using loss- and gain-of-function approaches, we demonstrated the role of ROR2 in the maintenance of flow-induced collective EC polarization. In vitro, laminar flow induced a relocalization of ROR2 at cell-cell junctions, associated with an interaction with VE-Cadherin and β-catenin. ROR2 expressed at the lateral pole of the cell induced adherens junctions remodeling in these areas. Regulation of EC polarization, as well as ROR2-induced junctional remodeling, were dependent on Cdc42 activation. These results demonstrated that the ROR2/PCP pathway regulates junctional remodeling at the cell pole and thus the collective polarization of endothelial cells.In a second part, we focused on EC responses to disturbed flow. Indeed, in vivo ROR2 was highly expressed in ECs of disturbed flow areas such as in the inner curvature of the aortic arch or in arterial bifurcations. We hypothesized that ROR2 expressed in these areas might control the inflammatory response and/or atherosclerotic plaque formation. In vitro, ROR2 loss of function induced a decrease in the expression of pro-inflammatory cytokines and adhesion molecules in ECs exposed to disturbed flow, suggesting a pro-inflammatory effect of ROR2. In vivo, deletion of Ror2 decreased leukocyte recruitment in the aortic arch of mice under physiological conditions but also in a mouse model of atherosclerosis (Double transgenic mice: ApoE-/-, Ror2iECKO). Thus, the endothelial expression of ROR2 in the aortic arch or in the bifurcations could activate the expression of pro-inflammatory genes (such as interleukin 1 and 8) and/or the expression of adhesion molecules to finally modify EC properties when they are subjected to disturbed flow.Together, these results showed that ROR2 is a new actor in the response of endothelial cells to blood flow by controlling cell polarity and the expression of genes involved in the maintenance of vascular integrity.Les cellules endothéliales (CE) tapissant les vaisseaux, perçoivent et traduisent les forces mécaniques du flux sanguin en signaux intracellulaires, et sont particulièrement sensibles aux forces de cisaillement. Cependant, en fonction des régions vasculaires les CE sont exposées à différents types de flux : principalement laminaire dans les régions droites de l’arbre vasculaire, et turbulent dans les régions de courbure ou de bifurcations, considéré comme athéroprone. Le but de cette thèse est d’étudier l’implication de ROR2, un acteur de la Polarité Cellulaire Planaire (PCP), dans la réponse vasculaire aux contraintes de cisaillement induites par le flux sanguin.Dans un premier temps, nous nous sommes focalisés sur les réponses des CE exposées à un flux laminaire. En effet dans les zones de flux laminaire les cellules s’allongent et se polarisent en fonction du flux, et présentent un phénotype athéroprotecteur. Par des approches in vivo, en utilisant des souris transgéniques délétées pour Ror2 spécifiquement dans les CE (Ror2iECKO), et par des approches in vitro de perte et gain de fonction, nous avons démontré l’importance de ROR2 dans le maintien de la polarisation collective des CE induite par le flux. In vitro, le flux laminaire induit une relocalisation de ROR2 au niveau des jonctions cellulaires, associée à une interaction avec la VE-Cadhérine et la β-caténine. ROR2 exprimé au niveau du pôle latéral de la cellule induit un remodelage des jonctions adhérentes. La régulation de la polarisation des CE, ainsi que le remodelage des jonctions induit par ROR2 sont dépendants de l’activation de Cdc42. Ces résultats démontrent que la voie ROR2/PCP régule le remodelage des jonctions cellulaires aux pôles de la cellule et ainsi la polarisation collective des cellules endothéliales.Dans une deuxième partie, nous nous sommes focalisés sur la réponse des cellules endothéliales dans un contexte de flux turbulent, athéroprone. En effet, ROR2 est fortement exprimé in vivo dans les CE exposées à un flux turbulent comme dans la courbure interne de la crosse aortique ou dans les bifurcations artérielles. Nous avons émis l’hypothèse que ROR2 exprimé dans ces zones pourrait contrôler la réponse inflammatoire et/ou la formation de la plaque d’athérome. In vitro, la perte de fonction de ROR2 induit une diminution de l’expression des cytokines pro-inflammatoires et des molécules d’adhésion dans les CE exposées au flux turbulent, suggérant un rôle pro-inflammatoire de ROR2. In vivo, la délétion de Ror2 diminue le recrutement des leucocytes dans la crosse aortique des souris en conditions physiologiques mais aussi dans un modèle murin d’athérosclérose (souris doublement transgénique: ApoE-/-, Ror2iECKO). Ainsi l’expression endothéliale de ROR2 dans la crosse aortique ou dans les bifurcations, pourrait activer l’expression de gènes pro-inflammatoires (comme les interleukines 1 et 8) et/ou modifier l’expression de molécules d’adhésion et finalement réguler les propriétés des CE lorsqu’elles sont soumises à un flux turbulent.L’ensemble de ces résultats a permis de montrer que ROR2 est un nouvel acteur de la réponse des cellules endothéliales au flux sanguin en contrôlant la polarité cellulaire, et l’expression de gènes impliqués dans le maintien de l’intégrité vasculaire