Growth of the extremophilic Deinococcus geothermalis DSM 11302 using co-substrate fed-batch culture

Deinococcus geothermalis metabolism has been scarcely studied to date, although new developments on its utilization for bioremediation have been carried out. So, largescale production of this strain and a better understanding of its physiology are required. A fed-batch experiment was conducted to achieve a high cell density non-limiting culture of D. geothermalis DSM 11302. A co-substrate nutritional strategy using glucose and yeast extract was carried out in a 20-L bioreactor in order to maintain a non-limited growth at a maximal growth rate of 1 h−1 at 45 °C. Substrate supplies were adjusted by monitoring online culture parameters and physiological data (dissolved oxygen, gas analyses, respiratory quotient, biomass concentration). The results showed that yeast extract could serve as both carbon and nitrogen sources, although glucose and ammonia were consumed too. Yeast extract carbon-specific uptake rate reached a value 4.5 times higher than glucose carbon-specific uptake rate. Cell concentration of 9.6 g L−1 dry cell weight corresponding to 99 g of biomass was obtained using glucose and yeast extract as carbon and nitrogen sources

Dynamique de la réponse physiologique d'Escherichia coli à des perturbations maîtrisées de son environnement (vers le développement de nouveaux outils de changement d'échelle)

Les bioréacteurs de grandes dimensions, en raison de phénomènes de transfert limitant, sont le siège d hétérogénéités se traduisant par des gradients locaux de concentration et température. Les microorganismes circulant au sein de ces bioréacteurs subissent donc des fluctuations environnementales qui peuvent affecter leur comportement aux niveaux métaboliques et/ou moléculaires. La réponse microbienne est fonction de la nature, de l intensité, de la fréquence et de la durée de la perturbation. L objectif de ce travail est l étude quantitative de l impact de l intensité, la fréquence et l amplitude d un stress nutritionnel sur le comportement dynamique d Escherichia coli, à savoir des ajouts pulsés de glucose lors de cultures continues en régime permanent. Un effort particulier est consacré au développement et à la validation des outils expérimentaux indispensables pour une caractérisation rigoureuse des dynamiques de réponses transitoires sur des échelles de temps allant de secondes à quelques minutes. Pour permettre le suivi in situ et en temps réel des changements métaboliques et moléculaires, une souche bioluminescente est mise en œuvre. Les réponses transitoires sont caractérisées par les vitesses spécifiques, les rendements, les profils d induction transcriptionnelle, les temps caractéristiques. Selon les différents scenarii réalisés, l ajustement du métabolisme face aux hétérogénéités de substrat est quantifié selon des échelles de temps aux niveaux macroscopiques et/ou moléculaires ; ces résultats originaux contribuent ainsi à l implémentation des connaissances sur les interactions dynamiques entre les phénomènes biologiques et les phénomènes physiques ; l enjeu réside à terme en l amélioration des processus d optimisation et d extrapolation des bioprocédés par l identification et la quantification des dynamiques des phénomènes limitantsIneffective mixing entailing heterogeneity issues within industrial bioreactors have been reported to affect microbial metabolisms at cellular and/or molecular levels. Substrate gradients inside large-scale bioreactors are common environmental fluctuations that microorganisms would have to encouter along with the bioprocess. Depending on intensity, frequency and duration of those fluctuations, microorganisms may respond in a different manner. The objective of this work is to study the impact of intensity, frequency and amplitude of glucose perturbations on the dynamics of Escherichia coli responses. An E. coli bioluminescent strain is used for in situ and real-time monitoring of both metabolic and transcriptional changes. For this purpose, short-term glucose excess was simulated, using pulse-based experiments into glucose-limited chemostat cultures. In addition, an important effort is devoted to the development and validation of technical and mathematical tools in order to acquire quantitative and kinetic data on time scales from seconds to minutes. The transient responses are characterized, using specific rates, yields, transcriptional induction profiles and characteristic response times, and are compared in the different defined perturbation scenarios. The results reflected the fact that short-term heterogeneities of substrate affect both cell metabolism and regulation at macroscopic and/or molecular levels. Quantitative understandings of the dynamics during transient responses to environmental perturbations can thus shed light on the bioprocess optimizationTOULOUSE-INSA-Bib. electronique (315559905) / SudocSudocFranceF

Quantitative Characterization of the Growth of Deinococcus geothermalis DSM-11302: Effect of Inoculum Size, Growth Medium and Culture Conditions

Due to their remarkable resistance to extreme conditions, Deinococcaceae strains are of great interest to biotechnological prospects. However, the physiology of the extremophile strain Deinococcus geothermalis has scarcely been studied and is not well understood. The physiological behaviour was then studied in well-controlled conditions in flask and bioreactor cultures. The growth of D. geothermalis type strains was compared. Among the strains tested, the strain from the German Collection of Microorganisms (Deutsche Sammlung von Mikroorganismen DSM) DSM-11302 was found to give the highest biomass concentration and growth rate: in a complex medium with glucose, the growth rate reached 0.75 h−1 at 45 °C. Yeast extract concentration in the medium had significant constitutive and catalytic effects. Furthermore, the results showed that the physiological descriptors were not affected by the inoculum preparation steps. A batch culture of D. geothermalis DSM-11302 on defined medium was carried out: cells grew exponentially with a maximal growth rate of 0.28 h−1 and D. geothermalis DSM-11302 biomass reached 1.4 g•L −1 in 20 h. Then, 1.4 gDryCellWeight of biomass (X) was obtained from 5.6 g glucose (Glc) consumed as carbon source, corresponding to a yield of 0.3 CmolX•CmolGlc−1 ; cell specific oxygen uptake and carbon dioxide production rates reached 216 and 226 mmol.CmolX −1•h −1 , respectively, and the respiratory quotient (QR) value varied from 1.1 to 1.7. This is the first time that kinetic parameters and yields are reported for D. geothermalis DSM-11302 grown on a mineral medium in well-controlled batch cultur

Experimental and statistical analysis of nutritional requirements for the growth of the extremophile Deinococcus geothermalis DSM 11300

Few studies concerning the nutritional requirements of Deinococcus geothermalis DSM 11300 have been conducted to date. Three defined media compositions have been published for the growth of this strain but they were found to be inadequate to achieve growth without limitation. Furthermore, growth curves, biomass concentration and growth rates were generally not available. Analysis in Principal Components was used in this work to compare and consequently to highlight the main compounds which differ between published chemically defined media. When available, biomass concentration, and/or growth rate were superimposed to the PCA analysis. The formulations of the media were collected from existing literature; media compositions designed for the growth of several strains of Deinococcaceae or Micrococcaceae were included. The results showed that a defined medium adapted from Holland et al. (Appl Microbiol Biotechnol 72:1074–1082, 2006) was the best basal medium and was chosen for further studies. A growth rate of 0.03 h-1 and a final OD600nm of 0.55 were obtained, but the growth was linear. Then, the effects of several medium components on oxygen uptake and biomass production by Deinococcus geothermalis DSM 11300 were studied using a respirometry-based method, to search for the nutritional limitation. The results revealed that the whole yeast extract in the medium with glucose is necessary to obtain a non-limiting growth of Deinococcus geothermalis DSM 11300 at a maximum growth rate of 0.64 h-1 at 45 °C

Optical Methods and Their Limitation to Characterize the Morphology and Granulometry of Complex Shape Biological Materials

Background and aim: Particle size and morphology of biomass (microorganism, lignocellulosic substrates) stand out as the major determinants of the bioprocess efficiency. Through its impact on rheology, it affects momentum, heat and and mass transfers within the bioreactor. Various techniques are available to characterize in-situ and ex-situ size and shape of particles. The most common methods are classified into three groups: (i) analysis of microscopic images; (ii) laser light diffraction and (iii) settling kinetics. In present work, five techniques are compared and discussed with model particles, microorganisms and lignocellulosic substrates. Methods: The used techniques aim to characterize size and shape (0.1 to 2000µm). In-situ and ex-situ measurements were used: chord length measurement (FBRM), diffraction light scattering (DLS), morpho-granulometry (MG), cytometry (CYT) and settling velocity (TUL). A set of height polystyrene microspheres (1.0 to 15.0µm) and microbeads (40 and 80µm) were used as reference. Yarrowia lipolytica is strictly aerobic yeast, belonging to the family of hemiascomycetes. Cells are subjected to mycelial transition induced by pH changes. Its morphology evolves from ovoid shape (5-7µm) up to filament. It was used to appreciate the ability to qualify and quantify filamentous shape (width, length). Finally, two cellulosic matrices, microcrystalline cellulose and coniferous paper pulp were selected to investigate complex fiber morphologies. Results: Specifications and limits of instruments are scrutinized. Sampling methods and preparation should be carefully considered. Optical measurements provide raw data (light intensity, frequency, images) from which morphological parameters will be straightly extracted or calculated based on assumptions (optical properties, particles geometry, theory). Considering diameters and associated number and volume distribution functions, techniques are compared with model calibrated microspheres. The mean values appear consistent between techniques but the magnitude of standard deviation extensively varies. Few instruments (MG, CYT) provide access to additional morphological criteria (length, width, aspect ratio). Mycelial kinetics and magnitude is accurately described by fiber length (MG). However a poor reliability of width (time of flight, CYT) is noticeable. Considering more complex lignocellulosic particles, the relative diameter values usually indicate similar trends whatever the techniques is. However, absolute values should be carefully considered and may deviated in large extend (5-10 times)

PRODUCTION D'EXOPOLYSACCHARIDES PAR PROPIONIBACTERIUM ACIDI-PROPIONICI SUR MICROFILTRAT DE LAIT. CINETIQUES DE FERMENTATION ET CARACTERISATION RHEOLOGIQUE DES POLYSACCHARIDES

NANCY/VANDOEUVRE-INPL (545472102) / SudocSudocFranceF

Applicability of GFP Microbial Whole Cell Biosensors to Bioreactor Operations : Mathematical Modeling and Related Experimental Tools

International audienc

Short-term dynamic behavior of Escherichia coli in response to successive glucose pulses on glucose-limited chemostat cultures

The effect of repeated glucose perturbations on dynamic behavior of Escherichia coli DPD2085, yciG::LuxCDABE reporter strain, was studied and characterized on a short-time scale using glucose-limited chemostat cultures at dilution rates close to 0.18 h(-1). The substrate disturbances were applied on independent steady-state cultures, firstly using a single glucose pulse under different aeration conditions and secondly using repeated glucose pulses under fully aerobic condition. The dynamic responses of E. coli to a single glucose pulse of different intensities (0.25 and 0.6 g L-1) were significantly similar at macroscopic level, revealing the independency of the macroscopic microbial behavior to the perturbation intensity in the range of tested glucose concentrations. The dynamic responses of E. coli to repeated glucose pulses to simulate fluctuating environments between glucose-limited and glucose-excess conditions were quantified; similar behavior regarding respiration and by-product formations was observed, except for the first perturbation denoted by an overshoot of the specific oxygen uptake rate in the first minutes after the pulse. In addition, transcriptional induction of yciG promoter gene involved in general stress response, sigma(S), was monitored through the bioluminescent E. coli strain. This study aims to provide and compare short-term quantitative kinetics data describing the dynamic behavior of E. coli facing repeated transient substrate conditions. (C) 2013 Elsevier B. V. All rights reserved

Plasmid expression level heterogeneity monitoring via heterologous eGFP production at the single-cell level in Cupriavidus necator

A methodology for plasmid expression level monitoring of eGFP expression suitable for dynamic processes was assessed during fermentation. This technique was based on the expression of a fluorescent biosensor (eGFP) encoded on a recombinant plasmid coupled to single-cell analysis. Fluorescence intensity at single-cell level was measured by flow cytometry. We demonstrated that promoter evaluation based on single-cell analysis versus classic global analysis brings valuable insights. Single-cell analysis pointed out the fact that intrinsic fluorescence increased with the strength of the promoter up to a threshold. Beyond that, cell permeability increases to excrete the fluorescent protein in the medium. The metabolic load due to the increase in the eGFP production in the case of strong constitutive promoters leads to slower growth kinetics compared with plasmid-free cells. With the strain Cupriavidus necator Re2133, growth rate losses were measured from 3% with the weak constitutive promoter P-lac to 56% with the strong constitutive promoter P-j5. Through this work, it seems crucial to find a compromise between the fluorescence intensity in single cells and the metabolic load; in our conditions, the best compromise found was the weak promoter P-lac. The plasmid expression level monitoring method was tested in the presence of a heterogeneous population induced by plasmid-curing methods. For all the identified subpopulations, the plasmid expression level heterogeneity was significantly detected at the level of fluorescence intensity in single cells. After cell sorting, growth rate and cultivability were assessed for each subpopulation. In conclusion, this eGFP biosensor makes it possible to follow the variations in the level of plasmid expression under conditions of population heterogeneity. Key Points center dot Development of a plasmid expression level monitoring method at the single-cell level by flow cytometry. center dot Promoter evaluation by single-cell analysis: cell heterogeneity and strain robustness. center dot Reporter system optimization for efficient subpopulation detection in pure cultures

Study of plasmid-based expression level heterogeneity under plasmid-curing like conditions in Cupriavidus necator

International audiencePlasmid expression level heterogeneity in Cupriavidus necator was studied in response to stringent culture conditions, supposed to enhance plasmid instability, through plasmid curing strategies. Two plasmid curing strategies were compared based on their efficiency at generating heterogeneity in batch: rifampicin addition and temperature increase. A temperature increase from 30 degrees to 37 degrees C was the most efficient plasmid curing strategy. To generate a heterogeneous population in terms of plasmid expression levels, successive batches at supra optimal culture temperature (i.e. 37 degrees C) were initially conducted. Three distinct fluorescent subpopulations P-0 (not fluorescent), P-1 (low fluorescence intensity, median = 1 10(3)) and P-2 (high fluorescence intensity, median = 6 10(3)) were obtained. From there, the chemostat culture was implemented to study the long-term stress response under well-controlled environment at defined dilution rates. For dilution rates comprised between 0.05 and 0.10 h(-1), the subpopulation P-2 (62% vs 90%) was favored compared to P1 cells (54% vs 1%), especially when growth rate increased. Our biosensor was efficient at discriminating subpopulation presenting different expression levels under stringent culture conditions. Plus, we showed that controlling growth kinetics had a stabilizing impact on plasmid expression levels, even under heterogeneous expression conditions