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Stirring in bioreactors: significant progress for the production of advanced biofuels using enzymatic processes

December 2017

The challenges associated with climate change require the development of an energy mix promoting low-carbon energies.

IFPEN is focusing its research efforts on several strategic themes, including that of biofuel production using non-food resources.

The production of lignocellulosic ethanol (or 2G ethanol) requires an enzymatic hydrolysis step in order to convert cellulose into glucose, which will then be transformed into ethanol via alcoholic fermentation. This step involves the use of enzymes but it represents one of the major economic challenges in the development of the overall process due to the high cost of the enzymes, known as cellulases.

Most industrial cellulases are produced by the filamentous fungus Trichoderma reesei, thanks to its high secretion capacity, enabling to reach concentrations higher than 100 g/L[1]. The standard production process developed at IFPEN is conducted in two steps, one in batch mode and the other in fed-batch mode:

  • The first step consists in cultivating the fungi in stirred, aerated reactors with a capacity of several hundred m3. Very intensive stirring is required during this step to facilitate the transfer of oxygen in the air to the microorganisms. The filamentous morphology of the fungus is the source of the non-Newtonian rheological properties* of the medium and limits this transfer in high concentrations[2]. This is because the viscosity increases as the fungus concentration increases, hence the need for a very high agitation power[3].
  • Once the medium contains a high enough concentration of filamentous biomass, a switch is made to fed-batch mode, with the introduction of an inducing sugar feed. This feed is limiting, i.e., it is below the maximum consumption capacity of the fungi and triggers a metabolic change. The production step follows, during which the fungi produce the target enzymes (cellulases)

The stirring technology employed in industrial reactors is critical for enzyme production since it has to deliver optimal conditions in terms of oxygen transfer and substrate concentration homogenization, while meeting a dual constraint: limiting energy consumption and minimizing the mechanical impact of shear on the microorganisms. This latter area has been the focus of major research efforts at IFPEN in recent years. This has been made possible by the development of innovative characterization tools, concerning the characterization of the rheology of media[4] and image processing for the morphology of filamentous fungi[5], as illustrated in figure 1.

These tools were applied to the characterization of fermentations performed in a broad variety of stirring conditions in terms of power supplied, type of stirring system and size or reactor. The wealth of data obtained - unique in the field - made it possible to identify relevant hydrodynamic descriptors to predict the rheology, morphology and fermentation performance of the filamentous fungus.

* The viscosity of which varies with shear rate.

Figure 1: Trichoderma reesei cultivation in a 3-L reactor and the image obtained from a microscopic analysis of the morphology of the fungi.

Compared with various criteria traditionally used to quantify shear, the most relevant criterion appears to be the EDCF-εmax, or Eddy Dissipation/Circulation Function criterion. This criterion is defined by the ratio between the maximum energy dissipation in the wake of the paddles of the agitation system (εmax) and the average time separating two circulations of fungi in the zone of highest dissipation (cycle time). The EDCF-εmax criterion is particularly useful for the large-scale extrapolation of fermentations studied in the laboratory since it provides excellent correlation with key quantities (rheology, morphology, fungus growth), thereby enabling a predictive approach regarding the quantitative performance of industrial fermenters. This is illustrated in figure 2, which presents the evolution of parameters of interest throughout the growth phase, with respect to the criterion selected.

                                              Click on the picture to enlarge it

Figure 2: Demonstration of the respective dependence of growth rate, viscosity and morphology with respect to the EDCF-εmax criterion.

During the enzyme production phase, a quantitative decrease in production was observed after excessive agitation. A proteomic analysis of the intracellular proteins at various agitation levels shows that the type of proteins synthesized is affected, with a reduction in the production of target cellulases and an increase in the synthesis of stress response proteins and those involved in central metabolism. This result suggests greater energy expenditure for cellular maintenance to the detriment of cellulase production[6].

Since the EDCF-εmax criterion can be easily linked to known agitation system properties, such as their intrinsic pumping and energy power capacity, it represents an invaluable aid in terms of the choice of future agitation technologies.

Much of the research was conducted in collaboration with Professors C. Béal of AgroParisTech and A.W. Nienow of the University of Birmingham, within the context of a thesis financed by the ADEME, carried out at IFPEN and defended in 2016[7].
Testimonies of the team of Nicolas Hardy's thesis (in French): 

The research was hailed by the scientific community: it scooped the prize for the best poster at the 2017 SFGP 2017[6] conference, in the "Bio-production innovations” category and illustrated the front cover of volume 173 of the Chemical Engineering Science journal[8, 9], published in December 2017.

Scientific contacts:   -



[1] Ben Chaabane F, Chaussepied B. Process for the continuous production of cellulases by a filamentous fungus using a carbon substrate obtained from an acid pretreatment.
>> US Patent 9249402 B2; 2016
[2] Gabelle J.C., Jourdier E., Licht R., Ben Chaabane F., Henaut I., Morchain J., Augier F., Impact of rheology on the mass transfer coefficient during the growth phase of Trichoderma reesei in stirred bioreactors, Chem.Eng.Sci, 75, 408-417, 2012.
>>DOI:1 0.1016/j.ces.2012.03.053
[3] Gabelle J.C., Augier F., Carvalho A., Rousset E., Morchain J., Effect of Tank Size on kLa and Mixing Time in Aerated Stirred Reactors With Non-Newtonian Fluids., Can.J.Chem.Eng., 89, 2011.
>> DOI: 10.1002/cjce.20571
[4] Hardy N., Henaut I., Augier F., Béal C., Ben Chaabane F., Rhéologie des champignons filamenteux : un outil pour la compréhension d’un procédé de production de biocatalyseurs utilisés pour la production de bioéthanol, Rhéologie, Vol. 27, 43-48, 2015.
[5] Hardy N., Moreaud M., Guillaume D., Augier F., Nienow A., Béal C., Ben Chaabane F., Advanced digital image analysis method dedicated to the characterization of the morphology of filamentous fungus, Journal of Microscopy, Vol. 266, Issue 2 2017, pp. 126-140.
>> DOI: 10.1111/jmi.12523
[6] Nicolas Hardy, Fadhel Ben Chaabane, Frédéric Augier, Alvin W. Nienow, Catherine Béal. Identification of fluid dynamic stress response of T. reesei during continuous fermentations conducted at high agitation rate. 16ème Congrès de la Société Française de Génie des Procédés (SFGP), Jul 2017, Nancy, France
>> HAL-01561070
[7] Nicolas Hardy. Thèse de Doctorat : Identification des critères d’extrapolation du procédé de production de cellulases par Trichoderma reesei en utilisant l’approche "scale-down", 2016
>> HAL-01637274
[8] Hardy N, Augier F, Nienow A. W., Béal C., Ben Chaabane F., Scale-up criteria for Trichoderma reesei fermentation, Chemical Engineering Science, 172, 2017, pp 158-168
>> DOI: 10.1016/j.ces.2017.06.034
[9] Cover page, Chem.Eng.Sci, Vol 173, Dec 2017
>> DOI: 10.1016/S0009-2509(17)30562-6

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