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)

Etude des dynamiques de réponses physiologiques et métaboliques de Yarrowia lipolytica à des perturbations environnementales physico-chimiques

Due to limited mixing capacities, heterogeneities regularly occur when scaling-up bioreactors for large-scale production. Microbial cultures are continuously exposed to local gradients in fundamental process parameters such as substrate, pH, temperature and dissolved oxygen DO concentration. These micro-environmental fluctuations may have detrimental effects on cellular growth, metabolism and morphology, depending on the nature, intensity, duration and/or frequency of the fluctuations encountered. The aim of this study was to investigate the impact of pH and DO fluctuations on the dynamic behavior of Yarrowia lipolytica, a microorganism with a promising biotechnological potential, at both morphological and metabolic levels. For this purpose, batch and continuous cultivations modes were preferentially adopted, as it enabled respectively, the study of the stress response of yeast populations growing at their maximum specific rate, and at various controlled specific growth rates in physiological steady-states. In addition, an important effort was devoted to the development and validation of morphological methods in order to acquire quantitative characterization of the response dynamics at the population scale. The macroscopic behavior of Y. lipolytica was assessed through examining the patterns of growth, viability, glucose uptake, oxygen consumption, organic acid and carbon dioxide production rates. Changes in the yeast morphology were characterized at the cell population level by means of flow cytometry, morphogranulometry and diffraction light scattering techniques. The results reflected no significant effect of pH and DO fluctuations on the macroscopic behavior (specific rates, yields, viability) of the yeast. Nevertheless, mycelial growth was induced upon exposure to both stressors, only in glucose-excess environments, suggesting therefore an impact of glucose levels on the regulation of dimorphic transition in Y. lipolytica. Controlling residual glucose concentrations in Y. lipolytica fermentations may contribute to a better monitoring of its morphological changes in response to environmental stimuli. Such data would help to optimize bioprocess performances at the industrial scale since it alleviates physico-chemical impacts due to filamentous cells.En raison des capacités de mélange limitantes, des hétérogénéités au sein des bioréacteurs se produisent régulièrement lors de l’extrapolation à l’échelle industrielle. En conséquence, les microorganismes circulant au sein de ces bioréacteurs sont continuellement exposés à des gradients locaux au niveau des paramètres fondamentaux du procédé tel que le pH, la température, la concentration en substrat et en oxygène dissous. Ces fluctuations micro-environnementales peuvent affecter la croissance, le métabolisme et la morphologie des cellules, en fonction de la nature, de l’intensité, de la durée et/ou de la fréquence de la perturbation rencontrée. L’objectif de ce travail est l’étude quantitative de l’impact des fluctuations de pH et d’oxygène dissous sur le comportement dynamique de Yarrowia lipolytica, une levure avec un potentiel biotechnologique prometteur, aussi bien aux niveaux morphologique que métabolique. Pour répondre à cet objectif, des cultures en bioréacteur en conditions d’environnement contrôlé ont été mises en œuvre afin d’établir un lien de causalité directe entre la perturbation et la réponse observée. L’implémentation de deux modes de cultures différents (batch et chemostat) a permis de caractériser le comportement dynamique des populations cellulaires dans des états physiologiques différents: En mode continu, toutes les cellules sont dans le même état physiologique et se multiplient à la même vitesse de croissance, tandis que des sous-populations de levures dans des états physiologiques distincts peuvent cohabiter dans les cultures en mode batch. Un effort important a été consacré au développement et validation des méthodes pour une quantification rigoureuse des évolutions morphologiques de Y. lipolytica à l’échelle de la population. Le comportement macroscopique de la levure a été caractérisé par l’évaluation des dynamiques de croissance, la viabilité, les vitesses de consommation du glucose et d’oxygène, ainsi que les vitesses de production d’acide organique et de dioxyde de carbone. Trois techniques, à savoir la cytométrie en flux (CYT), la morpho-granulométrie (MG) et la diffraction dynamique de la lumière (DLS) ont été employé pour la quantification du phénomène d’élongation. Les résultats obtenus démontrent qu’il n’y a pas d’effet significatif des fluctuations de pH et d’oxygène dissous sur le comportement macroscopique (vitesses spécifiques, rendements, viabilité) de la levure. Néanmoins, une transition micellaire a été induite en réponse aux deux facteurs de stress (pH and pO2) seulement en conditions d’excès de glucose, suggérant ainsi un impact de la concentration résiduelle de glucose sur la régulation de dimorphisme chez Y. lipolytica. Le contrôle et la régulation de la concentration de glucose dans le milieu peut contribuer à une meilleure maitrise des changements morphologiques de Y. lipolytica en réponse à des stimuli de l’environnement

Impacts of environmental conditions on product formation and morphology of Yarrowia lipolytica

The yeast Yarrowia lipolytica is an industrially important microorganism with distinctive physiological and metabolic characteristics. A variety of external factors (e.g., pH, temperature, and nutrient availability) influences the behavior of the yeast and may act as stress conditions which the cells must withstand and adapt. In this mini review, the impacts of environmental factors on the morphology and metabolite production by Y. lipolytica are summarized. In this regard, detailed insights into the effectors involved in the dimorphic transition of Y. lipolytica, the cultivation conditions employed, as well as the methods applied for the morphological characterization are highlighted. Concerning the metabolism products, a special focus is addressed on lipid and citric acid metabolites which have attracted significant attention in recent years. The dependence of lipid and citric acid productivity on key process parameters, such as media composition and physico-chemical variables, is thoroughly discussed. This review attempts to provide a recent update on the topic and will serve as a meaningful resource for researchers working in the field

Dynamic behavior of Yarrowia lipolytica to well-controlled pH and oxygen perturbations: dependence of the stress response on the culture mode

Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and physiological changes in response to environmental stressor

Comparison of optical methods to characterize morphology and size distribution of model particles and mycelial transition of Yarrowia lipolytica

In: 10th European Congress of Chemical Engineering; 3rd European Congress of Applied Biotechnology and 5th European Process Intensification Conference (ECCE10+ECAB3+EPIC5). Submission-2649. Topic : Biochemical Engineering / ECAB3 / Bioproducts (or bio-based products). Nice, France, September 27th - October 1st 2015In: 10th European Congress of Chemical Engineering; 3rd European Congress of Applied Biotechnology and 5th European Process Intensification Conference (ECCE10+ECAB3+EPIC5). Submission-2649. Topic : Biochemical Engineering / ECAB3 / Bioproducts (or bio-based products). Nice, France, September 27th - October 1st 2015Comparison of optical methods to characterize morphology and size distribution of model particles and mycelial transition of [i]Yarrowia lipolytica[/i] . 10. European Congress of Chemical Engineering - ECCE 201

Influence of oxygen availability on the metabolism and morphology of Yarrowia lipolytica: insights into the impact of glucose levels on dimorphism

Dynamic behavior of Yarrowia lipolytica W29 strain under conditions of fluctuating, low, and limited oxygen supply was characterized in batch and glucose-limited chemostat cultures. In batch cultures, transient oscillations between oxygen-rich and -deprived environments induced a slight citric acid accumulation (lower than 29 mg L−1). By contrast, no citric acid was detected in continuous fermentations for all stress conditions: full anoxia (zero pO2 value, 100% N2), limited (zero pO2 value, 75% of cell needs), and low (pO2 close to 2%) dissolved oxygen (DO) levels. The macroscopic behavior (kinetic parameters, yields, viability) of Y. lipolytica was not significantly affected by the exposure to DO fluctuations under both modes of culture. Nevertheless, conditions of oxygen limitation resulted in the destabilization of the glucose-limited growth during the continuous cultivations. Morphological responses of Y. lipolytica to DO oscillations were different between batch and chemostat runs. Indeed, a yeast-to-mycelium transition was induced and progressively intensified during the batch fermentations (filamentous subpopulation reaching 74% (v/v)). While, in chemostat bioreactors, the culture consisted mainly of yeast-like cells (mean diameter not exceeding 5.7 μm) with a normal size distribution. During the continuous cultures, growth at low DO concentration did not induce any changes in Y. lipolytica morphology. Dimorphism (up to 80.5% (v/v) of filaments) was only detected under conditions of oxygen limitation in the presence of a residual glucose excess (more than 0.75 g L−1). These data suggest an impact of glucose levels on the signaling pathways regulating dimorphic responses in Y. lipolytica

Accelerostat study in conventional and microfluidic bioreactors to assess the key role of residual glucose in the dimorphic transition of Yarrowia lipolytica in response to environmental stimuli

International audienceYarrowia lipolytica, with a diverse array of biotechnological applications, is able to grow as ovoid yeasts or filamentous hyphae depending on environmental conditions. This study has explored the relationship between residual glucose levels and dimorphism in Y. lipolytica. Under pH stress conditions, the morphological and physiological characteristics of the yeast were examined during well-controlled accelerostat cultures using both a 1 L-laboratory scale and a 1 mL-microfluidic bioreactor. The accelerostat mode, via a smooth increase of dilution rate (D), enabled the cell growth rate to increase gradually up to the cell wash-out (D >mu max of the strain), which was accompanied by a progressive increase in residual glucose concentration. The results showed that Y. lipolytica maintained an ovoid morphology when residual glucose concentration was below a threshold value of around 0.35-0.37 mg L-1. Transitions towards more elongated forms were triggered at this threshold and progressively intensified with the increase in residual glucose levels. The effect of cAMP on the dimorphic transition was assessed by the exogenous addition of cAMP and the quantification of its intracellular levels during the accelerostat. cAMP has been reported to be an important mediator of environmental stimuli that inhibit filamentous growth in Y. lipolytica by activating the cAMP-PKA regulatory pathway. It was confirmed that the exogenous addition of cAMP inhibited the mycelial morphology of Y. lipolytica, even with glucose concentrations exceeding the threshold level. The results suggest that dimorphic responses in Y. lipolytica are regulated by sugar signaling pathways, most likely via the cAMP-PKA dependent pathway

Dynamic behavior of Yarrowia lipolytica in response to pH perturbations: dependence of the stress response on the culture mode

Yarrowia lipolytica, a non-conventional yeast with a promising biotechnological potential, is able to undergo metabolic and morphological changes in response to environmental conditions. The effect of pH perturbations of different types (pulses, Heaviside) on the dynamic behavior of Y. lipolytica W29 strain was characterized under two modes of culture: batch and continuous. In batch cultures, different pH (4.5, 5.6 (optimal condition), and 7) were investigated in order to identify the pH inducing a stress response (metabolic and/or morphologic) in Y. lipolytica. Macroscopic behavior (kinetic parameters, yields, viability) of the yeast was slightly affected by pH. However, contrary to the culture at pH 5.6, a filamentous growth was induced in batch experiments at pH 4.5 and 7. Proportions of the filamentous subpopulation reached 84 and 93 % (v/v) under acidic and neutral conditions, respectively. Given the significant impact of neutral pH on morphology, pH perturbations from 5.6 to 7 were subsequently assayed in batch and continuous bioreactors. For both process modes, the growth dynamics remained fundamentally unaltered during exposure to stress. Nevertheless, morphological behavior of the yeast was dependent on the culture mode. Specifically, in batch bioreactors where cells proliferated at their maximum growth rate, mycelia were mainly formed. Whereas, in continuous cultures at controlled growth rates (from 0.03 to 0.20 h-1) even closed to the maximum growth rate of the stain (0.24 h-1), yeast-like forms predominated. This pointed out differences in the kinetic behavior of filamentous and yeast subpopulations, cell age distribution, and pH adaptive mechanisms between both modes of culture

Comparison of methods to explore the morphology and granulometry of biological particles with complex shapes: Interpretation and limitations

International audienc

Characterization of mycelial transition of Yarrowia lipolytica during oxidative cultures: comparison of optical methods and their limitation, impact of morphology on rheological behavior

In : 2nd Braunschweig International Symposium on Pharmaceutical Engineering Research SPhERe, TU Braunschweig, Germany September 06 to 08, 2017.Characterization of mycelial transition of Yarrowia lipolytica during oxidative cultures: comparison of optical methods and their limitation, impact of morphology on rheological behavior. 2. Braunschweig International Symposium on Pharmaceutical Engineering Research SPhER