Optimizing pressurized liquid extraction of microbial lipids using the response surface method

International audienceResponse surface methodology (RSM) was used for the determination of optimum extraction parameters to reach maximum lipid extraction yield with yeast. Total lipids were extracted from oleaginous yeast (Rhodotorula glutinis) using pressurized liquid extraction (PLE). The effects of extraction parameters on lipid extraction yield were studied by employing a second-order central composite design. The optimal condition was obtained as three cycles of 15 min at 100 °C with a ratio of 144 g of hydromatrix per 100 g of dry cell weight. Different analysis methods were used to compare the optimized PLE method with two conventional methods (Soxhlet and modification of Bligh and Dyer methods) under efficiency, selectivity and reproducibility criteria thanks to gravimetric analysis, GC with flame ionization detector, High Performance Liquid Chromatography linked to Evaporative Light Scattering Detector (HPLC-ELSD) and thin-layer chromatographic analysis. For each sample, the lipid extraction yield with optimized PLE was higher than those obtained with referenced methods (Soxhlet and Bligh and Dyer methods with, respectively, a recovery of 78% and 85% compared to PLE method). Moreover, the use of PLE led to major advantages such as an analysis time reduction by a factor of 10 and solvent quantity reduction by 70%, compared with traditional extraction methods

Towards a Microbial Production of Fatty Acids as Precursors of Biokerosene from Glucose and Xylose Vers une production microbienne d’acides gras en vue de l’application biokérosène à partir de glucose et xylose

The aviation industry considers the development of sustainable biofuels as one of the biggest challenges of the next ten years. The aim is to lower the environmental impact of the steadily increasing use of fossil fuels on climate change, yielding greater energy independence and fuel security. Thus, the development of a new route for the production of lipids from renewable non-food resources is now being promoted with the recent ASTM certification of hydrotreated oils. Our study focuses on the potential of growth of the oleaginous yeast Rhodotorula glutinis using glucose and xylose which can come from renewable lignocellulosic substrates and of lipid accumulation using glucose as substrate. Experiments were carried out in fed-batch mode which allowed feed flux management. Carbon fluxes were controlled with modifying xylose/glucose ratios to quantify metabolism in optimal growth condition. Besides, the management of carbon and nitrogen fluxes allowed characterizing lipid accumulation. Thus, it has been shown that the yeast Rhodotorula glutinis can simultaneously consume glucose and xylose. When the ratio xylose/glucose increased, the growth rate and the carbon conversion yield into biomass decreased: it was of 0.36 h-1 and 0.64 Cmol x*.Cmol glu-1 for pure glucose, it was of 0.15 h-1 and 0.56 Cmol.Cmol-1 for 10% xylose and it was of 0.037 h-1 and 0.18 Cmol.Cmol-1 for pure xylose. The necessity to maintain residual growth and to manage carbon fluxes to optimize lipid accumulation performance was revealed. Lipid accumulation on glucose engendered a final biomass concentration of 150 gCDW.L-1, microbial production (72% of lipids) and maximal productivity over 1.48 glip.L-1.h-1. The culture temperature is an important parameter to modulate the lipid profile. The results were encouraging. Lipid accumulation using lignocellulosic feedstock was shown to be a highly promising route. <br> Le développement de filières de production de molécules énergétiques en substitution au kérosène constitue un défi majeur pour l’industrie aéronautique afin de minimiser l’impact environnemental de son activité et de répondre à ses besoins en énergie, dont la demande est croissante. Le développement d’une nouvelle voie de production de lipides à partir de ressources renouvelables non alimentaires ouvre des perspectives prometteuses avec la certification ASTM des huiles hydrotraitées. Les travaux expérimentaux consistent en l’étude, d’une part, des potentialités de croissance de la levure oléagineuse Rhodotorula glutinis à partir de glucose et/ou xylose, substrats osidiques issus des ressources lignocellulosiques, et d’autre part, des potentialités d’accumulation de lipides à partir de glucose. Des cultures en mode fed-batch ont permis le contrôle des flux d’alimentation : en carbone, en condition de croissance, selon un ratio xylose/glucose variable pour la quantification du métabolisme, et en azote, en condition de production de lipides. Il a été montré que la levure Rhodotorula glutinis est capable de consommer simultanément le glucose et le xylose. Le taux de croissance et le rendement de conversion du carbone en biomasse diminuent en fonction de la composition du mélange xylose/glucose : à savoir 0,36 h-1 et 0,43 Cmol. x*.Cmol glu-1 sur glucose pur, 0,15 h-1 et 0,56 Cmol.Cmol-1 sur 10 % de xylose, 0,037 h-1 et 0,18 Cmol.Cmol-1 sur xylose pur. Par ailleurs, lors d’expérimentation en condition. d’accumulation lipidique, il a été mis en évidence la nécessité de maintenir une croissance résiduelle par le contrôle des flux d’azote et de carbone. Lors de la phase de production de lipides sur glucose, il a été ainsi obtenu une concentration finale en biomasse de 150 gCDW.L-1 contenant 72 % de lipides en masse; la productivité volumétrique maximale atteint 1,5 glip.L-1.h-1, avec un rendement de conversion du glucose en lipides égal à 95 % du rendement théorique limite La température de culture se révèle un paramètre opératoire important pour la modulation du profil lipidique. Ces résultats sont originaux et ils ont permis l’obtention de très hautes performances, en culture intensive. Ils argumentent des potentialités de développement d’une stratégie de production de lipides par conversion de toutes les fractions osidiques des substrats lignocellulosiques pour des usages biokérosène après hydrotraitement

Soft-Sensors for Monitoring B. Thuringiensis Bioproduction

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Modélisation hybride et dynamique de la production d'un nanobody anti-venin (de scorpion) dans E. coli CH10-12 et E. coli NbF12-10

International audienceImmunotherapy is a specific treatment for scorpion stings, with antibody fragments being used to neutralize scorpion neurotoxins. Nanobodies (VHH), fragments of camelid antibodies, were successfully produced intracellularly in Escherichia coli WK6 in fedbatch cultures. Their production was further enhanced by dynamic modelling. Two dynamic modelling approaches to describe nanobody CH12-10 production as a function of the induction temperature are proposed in this work. The first one is a kinetic model and the second is a hybrid approach, coupling a mass balance with support vector machine (SVM). Both models were calibrated and validated with independent data sets. Results reveal that the hybrid model procures better predictions than the kinetic model. Finally, the hybrid model was improved by retraining the SVM Model, resulting in a Normed Root Mean Square Error (NRMSE) values between 0.1148 and 0.8523

Optimization of lipid extraction from the oleaginous yeasts Rhodotorula glutinis and Lipomyces kononenkoae

The constant growing demand for vegetable oil for biodiesel and food is raising many environmental concerns about the sustainability of its production based on crops. Oleaginous yeasts show great potential to end with those concerns due to their high lipid productivity in small areas. To evaluate their productivity in lipids, an efficient and reproducible extraction process should be used. As no standard extraction process is available for the extraction of yeast lipids, an optimized extraction process is presented. In this work, the lipids extraction process for the yeasts Rhodotorula glutinis and Lipomyces kononenkoae is optimized using bead beating for cell rupture and introducing adaptations of the two most used extraction methods (Bligh and Dyer and Folch). For Rhodotorula g. the optimum extraction conditions are obtained by the Bligh and Dyer method applying 4.8 cycles of 47 s with 0.7 g of glass beads. For Lipomyces k. the optimum extraction conditions make use of the Folch method applying seven cycles of 42 s with 0.54 g of glass beads. These results reinforce the idea that, for each yeast, different extraction processes may be needed to correctly determine the lipid yield. The extraction procedure was further evaluated with less harmful solvents. Toluene was tested as a possible substitute of chloroform, and ethanol as a possible substitute of methanol. With the optimized extraction process, better results for Lipomyces k. were obtained using toluene and ethanol, while for Rhodotorula g. toluene proved to be a valid substitute of chloroform but ethanol is far less effective than methanol.This study was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), FCT Doctoral grant (SFRH/BD/80490/2011) attributed to Bruno Vasconcelos and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio