Application of the sale‐down methodology to study the effect of mixing on Trichoderma reesei physiology and enzyme production

International audienceIn this study, the effect of oxygen oscillations on enzyme production has been investigated during cultures of T. reesei. By using 2 bioreactors a decrease of 20% in the specific production rate and yield for long-time cycling exposure to anaerobic conditions (15 min) was observed, whereas a short cycle of anaerobiosis (3-4 min) was sufficient by using 1 bioreactor. Our results indicate the sensitivity of T. reesei to anaerobiosis and the fact that the metabolic response is complex and not immediate because it depends on the distribution of the residence times of the microorganisms in the aerated or non‐aerated zones

Cellulase activity mapping of Trichoderma reesei cultivated in sugar mixtures under fed-batch conditions

International audienceBackground: On-site cellulase production using locally available lignocellulosic biomass (LCB) is essential for cost-effective production of 2nd-generation biofuels. Cellulolytic enzymes (cellulases and hemicellulases) must be produced in fed-batch mode in order to obtain high productivity and yield. To date, the impact of the sugar composition of LCB hydrolysates on cellulolytic enzyme secretion has not been thoroughly investigated in industrial conditions. Results: The effect of sugar mixtures (glucose, xylose, inducer) on the secretion of cellulolytic enzymes by a glucose-derepressed and cellulase-hyperproducing mutant strain of Trichoderma reesei (strain CL847) was studied using a small-scale protocol representative of the industrial conditions. Since production of cellulolytic enzymes is inducible by either lactose or cellobiose, two parallel mixture designs were performed separately. No significant difference between inducers was observed on cellulase secretion performance, probably because a common induction mechanism occurred under carbon flux limitation. The characteristics of the enzymatic cocktails did not correlate with productivity, but instead were rather dependent on the substrate composition. Increasing xylose content in the feed had the strongest impact. It decreased by 2-fold cellulase, endoglucanase, and cellobiohydrolase activities and by 4-fold β-glucosidase activity. In contrast, xylanase activity was increased 6-fold. Accordingly, simultaneous high β-glucosidase and xylanase activities in the enzymatic cocktails seemed to be incompatible. The variations in enzymatic activity were modelled and validated with four fed-batch cultures performed in bioreactors. The overall enzyme production was maintained at its highest level when substituting up to 75% of the inducer with non-inducing sugars. Conclusions: The sugar substrate composition strongly influenced the composition of the cellulolytic cocktail secreted by T. reesei in fed-batch mode. Modelling can be used to predict cellulolytic activity based on the sugar composition of the culture-feeding solution, or to fine tune the substrate composition in order to produce a desired enzymatic cocktail

Mitigation of hydrodynamics related stress in bioreactors: a key for the scale-up of enzyme production by filamentous fungi

International audienceDifferent impellers and stirring conditions are compared at the bench scale in order to identify the relevant hydrodynamic stress parameter that correlates best with the process characteristics, morphology, broth rheology and growth rate as measured during the culture of the filamentous fungi, T. reesei, used for the production of cellulases. A modified Energy Dissipation Circulation Function (EDCF) was the most relevant stress parameter, and its use is validated to predict fairly accurately the same process characteristics in an industrial bioreactor, as well as enzyme production. The results are of great importance for the scale-up of cellulase production for bioethanol manufacture from lignocellulosic biomass

7th Drug hypersensitivity meeting: part two

No abstract availabl

Intensification de la production d'éthanol biocarburant dans un bioréacteur bi-étagé avec recyclage cellulaire (modélisation et stratégie de conduite)

TOULOUSE-INSA (315552106) / SudocSudocFranceF

A new stoichiometric miniaturization strategy for screening of industrial microbial strains: application to cellulase hyper-producing <it>Trichoderma reesei</it> strains

<p>Abstract</p> <p>Background</p> <p>During bioprocess development, secondary screening is a key step at the boundary between laboratory and industrial conditions. To ensure an effective high-throughput screening, miniaturized laboratory conditions must mimic industrial conditions, especially for oxygen transfer, feeding capacity and pH stabilization.</p> <p>Results</p> <p>A feeding strategy has been applied to develop a simple screening procedure, in which a stoichiometric study is combined with a standard miniaturization procedure. Actually, the knowledge of all nutriments and base or acid requirements leads to a great simplification of pH stabilization issue of miniaturized fed-batch cultures. Applied to cellulase production by <it>Trichoderma reesei</it>, this strategy resulted in a stoichiometric mixed feed of carbon and nitrogen sources. While keeping the pH between shake flask and stirred bioreactor comparable, the developed shake flask protocol reproduced the strain behaviour under stirred bioreactor conditions. Compared to a an already existing miniaturized shake flasks protocol, the cellulase concentration was increased 5-fold, reaching about 10 g L^-1. Applied to the secondary screening of several clones, the newly developed protocol succeeded in selecting a clone with a high industrial potential.</p> <p>Conclusions</p> <p>The understanding of a bioprocess stoichiometry contributed to define a simpler and more effective miniaturization. The suggested strategy can potentially be applied to other fed-batch processes, for the screening of either strain collections or experimental conditions.</p

The impact of fluid-dynamic stress in stirred tank bioreactors on the synthesis of cellulases by Trichoderma reesei at the intracellular and extracellular levels

International audienceCellulases for bioethanol production are mainly made by fed-batch fermentation using a filamentous fungus, Trichoderma reesei. Agitation at different scales impacts on morphology, rheology and growth rate and can be correlated by EDCFεmax. Typically, EDCFεmax is much smaller at commercial scale and fungal size, viscosity and growth rate are greater. Here, to increase understanding, continuous culture in 3 L bioreactors using two EDCFεmax values were undertaken. The higher EDCFεmax decreased the cellulase production (concentration, 21%; specific production rate, 24%; protein yield, 20%) whilst proteomic analysis showed, at an intracellular level, a decrease of cellulase and hemicellulase synthesis. An increase of stress proteins also occurred, which may help cells to limit the impact of fluid dynamic stress. Also, cellulase production during continuous culture at the bench varied with EDCFεmax similarly to that between bench and commercial scale during fed-batch culture

Advanced digital image analysis method dedicated to the characterization of the morphology of filamentous fungus

International audienceFilamentous fungi have a complex morphology that induces fermentation process development issues, as a consequence of viscosity increase and diffusion limitations. In order to better understand the relationship between viscosity changes and fungus morphology during fermentations of Trichoderma reesei, an accurate image analysis method has been developed to provide quantitative and representative data for morphological analysis. This method consisted of a new algorithm called FACE that allowed sharp images to be created at all positions, segmentation of fungus, and morphological analysis using skeleton and topological approaches. It was applied and validated by characterizing samples of an industrial strain of Trichoderma reesei that had or had not been exposed to an extreme shear stress. This method allowed many morphological characteristics to be identified, among which nine relevant criteria were extracted, regarding the impact of shear stress on the fungus and on the viscosity of the fermentation medium