Fadhel Ben Chaabane - Academia.edu (original) (raw)

Papers by Fadhel Ben Chaabane

Cellulases for bioethanol production are mainly made by fed-batch fermentation using a filamentou... more Cellulases 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 EDCFemax. Typically, EDCFemax 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 EDCFemax values were undertaken. The higher EDCFemax 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 EDCFemax similarly to that between bench and commercial scale during fed-batch culture.

<p>Butanol and Isopropanol are naturally produced by the bacteria C. beijerinckii. ... more <p>Butanol and Isopropanol are naturally produced by the bacteria C. beijerinckii. Those products are used in large field of applications such as fuel and bulk chemicals. Since butanol is toxic at small concentration for cells, bacterial growth and metabolism are inhibited during classical batch fermentation (1). These phenomena lead to the production of low solvent concentration (around 7 g.L<sup>-1</sup>) and a low volumetric productivity (0,13 g.L<sup>-1</sup>.h<sup>-1</sup>) (2). Continuous fermentation can be performed in order to avoid product inhibition by  a continuous removal of fermentation broth. However, the solvent productive biomass is easily washout at high dilution rate because of the low maximum growth rate of the strain in this metabolism phase  (0,05 h<sup>-1</sup>) (3). To overcome this issue, cell immobilization of  C. beijerinckii by biofilm formation on solid support is the best solution. As a result, the biomass residence time can be uncorrelated from the hydraulic residence time leading to a higher viable biomass concentration in the bioreactor and consequently a higher volumetric productivity (up to 5 g.L<sup>-1</sup>.h-1 ) (4). Our study aimed  at evaluating biofilm viability which is an important parameter that is linked to process productivity and has been little studied in the case of the IBE fermentation (5).</p> <p>In this study we developed two techniques to monitor biofilm viability during immobilized cell fermentation: Flow cytometry (FC) and PMA qPCR. After FC analysis, a high background noise due to the biofilm extra polymeric substance is obtained. Consequently, an enzymatic  sequential enzymatic biofilm deconstruction using Dnase I and Proteinase K was developed . This pre-treatment successfully lowered the background noise of this analysis. The suspensions obtained were stained with carboxyfluoresceine diacetate (cFDA) and propidium iodide (PI) which are indicators of cellular activity and alteration of membrane integrity, respectively,  and analyzed by flow cytometry. The percentage of viable cells obtained after pre-treatment compared to the control sample is increased from 2.6 ± 0.9 % to 22.8 ± 8.6% because of the background noise decrease. PMA-qPCR confirmed the results obtained by flow cytometry without using enzymatic pre-treatment. Although FC is less accurate than PMA-qPCR, this technique is less time-consuming, cheaper and reliable to study biofilm viability.</p> <p><strong>References</strong></p> <ol> <li>Jones et al (1986) Acetone-Butanol Fermentation Revisited, Microbiological Reviews 50, 484–524.</li> <li>Ferreira dos Santos Vieira, C., Maugeri Filho, F., Maciel Filho, R., and Pinto Mariano, A. (2019) Isopropanol-butanol-ethanol (IBE) production in repeated-batch cultivation of Clostridium beijerinckii DSM 6423 immobilized on sugarcane bagasse, Fuel, 116708.</li> <li>Ahmed, I., Ross, R. A., Mathur, V. K., and Chesbro, W. R. (1988) Growth rate dependence of solventogenesis and solvents produced by Clostridium beijerinckii, Appl Microbiol Biotechnol 28, 182–187.</li> <li>Survase, S. A., van Heiningen, A., and Granström, T. (2013) Wood pulp as an immobilization matrix for the continuous production of isopropanol and butanol, J. Ind. Microbiol. Biotechnol. 40, 209–215.</li> <li>Qureshi, N., Lai, L. L., and Blaschek, H. P. (2004) Scale-Up of a High Productivity Continuous Biofilm Reactor to Produce Butanol by Adsorbed Cells of Clostridium Beijerinckii, Featuring Tissue Engineering 82, 164–173.</li> </ol>

Chemical Engineering Science

Scale-up of aerobic fungal fermentation processes still remains a challenging issue for the biote... more Scale-up of aerobic fungal fermentation processes still remains a challenging issue for the biotechnology industry. This difficulty arises due to the complex interactions between operating conditions (agitation, aeration, etc.), the physicochemical state of the broth (viscosity, the dissolved oxygen concentration, etc.) and the biology of fungi (growth, production, morphology, etc.). Because of their size, filamentous fungi are affected by fluid dynamic stresses but quantification of this complex parameter is a difficult task. In general, indirect criteria are used for the effect of fluid dynamic stresses on scale-up (tip speed, power draw or the energy dissipation/circulation function (EDCF)). In order to better understand the impact of such criteria on the fermentation of the fungus Trichoderma reesei, a wide range of agitation conditions has been explored. The morphology of T. reesei fungus, its specific growth rate and the rheological properties of the broth have all been measured both at bench 2 scale (~ 2.5 L) and for the first time, at a typical commercial scale. These three aspects of the fermentation at both scales were then compared with respect to tip speed, specific power and EDCF. This work has shown that tip speed as a correlator of any of these parameters is totally ineffective whilst the EDCF is clearly the best for extrapolating laboratory data to the commercial scale.

Bioprocess and Biosystems Engineering, 2006

The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially ca... more The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially capable of giving very high ethanol productivity-was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30 degrees Celsius and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l(-1) and ethanol concentrations of 31 and 65 g l(-1) were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l(-1) in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l(-1) h(-1)) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3 degrees GL) in the two-stage system.

Bioprocess and Biosystems Engineering, 2006

The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially ca... more The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially capable of giving very high ethanol productivity-was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30°C and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l À1 and ethanol concentrations of 31 and 65 g l À1 were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l À1 in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l À1 h À1 ) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3°GL) in the two-stage system.

Oil & Gas Science and Technology – Revue d’IFP Energies nouvelles, 2013

and available online here Cet article fait partie du dossier thématique ci-dessous publié dans la... more and available online here Cet article fait partie du dossier thématique ci-dessous publié dans la revue OGST, Vol. 68, n°4, pp. 621-783 et téléchargeable ici D o s s i e r DOSSIER Edited by/Sous la direction de : A. Daudin et A. Quignard

L’objectif est l’intensification de la production d’éthanol biocarburant par Saccharomyces cerevi... more L’objectif est l’intensification de la production d’éthanol biocarburant par Saccharomyces cerevisiae dans un Bioréacteur Bi-étagé avec Recyclage Cellulaire (BBRC) en mode continu. A cette fin, nous avons modélisé le fonctionnement du bioréacteur et le comportement dynamique de la levure. Le modèle développé associe les équations de bilans matière et les cinétiques microbiennes en distinguant la biomasse totale et viable en fonction des états physiologiques de la levure dans les deux étages. Le principe du BBRC est l’utilisation du potentiel de la levure à son niveau optimal ; il combine le découplage spatial croissance/production d’éthanol, l’obtention de hautes densités cellulaires et la gestion de l’activité cellulaire par le recyclage de la biomasse entre les deux étages. Des cultures ont permis la quantification du comportement de la levure dans chaque étage, en régimes transitoires et permanents, pour différentes conditions opératoires. La confrontation théorie-expérience est ...

AMB Express, 2016

Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperp... more Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery.

: Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck ... more : Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck of the production process of bioethanol from lignocellulose. It is generally admitted that the most efficient organism for the production of cellulolytic enzymes is the fungus Trichoderma reesei, mostly due to its high secretion capacity. Unfortunately, this fungus secretes very low concentrations of β-glucosidase, thereby often requiring β-glucosidase supplementation for complete cellulose hydrolysis. It is especially important to have sufficient quantities of β-glucosidase in order to prevent inhibition of cellobiohydrolases by cellobiose. In order to optimize the produced cocktail, a more efficient β-glucosidase was cloned into T. reesei CL847 strain. The new strain, called CL847 TR3002, secretes the evolved β-glucosidase and was tested for cellulase production in laboratory-scale reactors. Its growth kinetics and cellulase production were characterized using fed-batch and chemostat m...

A simple kinetic model of cellulase production by Trichoderma reesei on lactose was developed and... more A simple kinetic model of cellulase production by Trichoderma reesei on lactose was developed and parameters identified using bibliographic and personal data. Assuming a mean representation of industrial hyperproducer strains, this model was used to simulate and compare several cultivation strategies regarding three criteria: in addition to usual productivity and yield calculation, oxygen demand was studied and used as a constraint for protocol design. Results showed that none of the protocols maximizes both productivity and yield, requiring to find a compromise for these parameters. Moreover, substrate concentration in the feed was the main criterion for the choice of the protocol, while oxygen demand and biomass concentration were the main issues to reach high productivities. The model will be useful for an economic study of cultivation strategies.

Cellulases for bioethanol production are mainly made by fed-batch fermentation using a filamentou... more Cellulases 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 EDCFemax. Typically, EDCFemax 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 EDCFemax values were undertaken. The higher EDCFemax 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 EDCFemax similarly to that between bench and commercial scale during fed-batch culture.

<p>Butanol and Isopropanol are naturally produced by the bacteria C. beijerinckii. ... more <p>Butanol and Isopropanol are naturally produced by the bacteria C. beijerinckii. Those products are used in large field of applications such as fuel and bulk chemicals. Since butanol is toxic at small concentration for cells, bacterial growth and metabolism are inhibited during classical batch fermentation (1). These phenomena lead to the production of low solvent concentration (around 7 g.L<sup>-1</sup>) and a low volumetric productivity (0,13 g.L<sup>-1</sup>.h<sup>-1</sup>) (2). Continuous fermentation can be performed in order to avoid product inhibition by  a continuous removal of fermentation broth. However, the solvent productive biomass is easily washout at high dilution rate because of the low maximum growth rate of the strain in this metabolism phase  (0,05 h<sup>-1</sup>) (3). To overcome this issue, cell immobilization of  C. beijerinckii by biofilm formation on solid support is the best solution. As a result, the biomass residence time can be uncorrelated from the hydraulic residence time leading to a higher viable biomass concentration in the bioreactor and consequently a higher volumetric productivity (up to 5 g.L<sup>-1</sup>.h-1 ) (4). Our study aimed  at evaluating biofilm viability which is an important parameter that is linked to process productivity and has been little studied in the case of the IBE fermentation (5).</p> <p>In this study we developed two techniques to monitor biofilm viability during immobilized cell fermentation: Flow cytometry (FC) and PMA qPCR. After FC analysis, a high background noise due to the biofilm extra polymeric substance is obtained. Consequently, an enzymatic  sequential enzymatic biofilm deconstruction using Dnase I and Proteinase K was developed . This pre-treatment successfully lowered the background noise of this analysis. The suspensions obtained were stained with carboxyfluoresceine diacetate (cFDA) and propidium iodide (PI) which are indicators of cellular activity and alteration of membrane integrity, respectively,  and analyzed by flow cytometry. The percentage of viable cells obtained after pre-treatment compared to the control sample is increased from 2.6 ± 0.9 % to 22.8 ± 8.6% because of the background noise decrease. PMA-qPCR confirmed the results obtained by flow cytometry without using enzymatic pre-treatment. Although FC is less accurate than PMA-qPCR, this technique is less time-consuming, cheaper and reliable to study biofilm viability.</p> <p><strong>References</strong></p> <ol> <li>Jones et al (1986) Acetone-Butanol Fermentation Revisited, Microbiological Reviews 50, 484–524.</li> <li>Ferreira dos Santos Vieira, C., Maugeri Filho, F., Maciel Filho, R., and Pinto Mariano, A. (2019) Isopropanol-butanol-ethanol (IBE) production in repeated-batch cultivation of Clostridium beijerinckii DSM 6423 immobilized on sugarcane bagasse, Fuel, 116708.</li> <li>Ahmed, I., Ross, R. A., Mathur, V. K., and Chesbro, W. R. (1988) Growth rate dependence of solventogenesis and solvents produced by Clostridium beijerinckii, Appl Microbiol Biotechnol 28, 182–187.</li> <li>Survase, S. A., van Heiningen, A., and Granström, T. (2013) Wood pulp as an immobilization matrix for the continuous production of isopropanol and butanol, J. Ind. Microbiol. Biotechnol. 40, 209–215.</li> <li>Qureshi, N., Lai, L. L., and Blaschek, H. P. (2004) Scale-Up of a High Productivity Continuous Biofilm Reactor to Produce Butanol by Adsorbed Cells of Clostridium Beijerinckii, Featuring Tissue Engineering 82, 164–173.</li> </ol>

Chemical Engineering Science

Scale-up of aerobic fungal fermentation processes still remains a challenging issue for the biote... more Scale-up of aerobic fungal fermentation processes still remains a challenging issue for the biotechnology industry. This difficulty arises due to the complex interactions between operating conditions (agitation, aeration, etc.), the physicochemical state of the broth (viscosity, the dissolved oxygen concentration, etc.) and the biology of fungi (growth, production, morphology, etc.). Because of their size, filamentous fungi are affected by fluid dynamic stresses but quantification of this complex parameter is a difficult task. In general, indirect criteria are used for the effect of fluid dynamic stresses on scale-up (tip speed, power draw or the energy dissipation/circulation function (EDCF)). In order to better understand the impact of such criteria on the fermentation of the fungus Trichoderma reesei, a wide range of agitation conditions has been explored. The morphology of T. reesei fungus, its specific growth rate and the rheological properties of the broth have all been measured both at bench 2 scale (~ 2.5 L) and for the first time, at a typical commercial scale. These three aspects of the fermentation at both scales were then compared with respect to tip speed, specific power and EDCF. This work has shown that tip speed as a correlator of any of these parameters is totally ineffective whilst the EDCF is clearly the best for extrapolating laboratory data to the commercial scale.

Bioprocess and Biosystems Engineering, 2006

The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially ca... more The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially capable of giving very high ethanol productivity-was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30 degrees Celsius and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l(-1) and ethanol concentrations of 31 and 65 g l(-1) were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l(-1) in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l(-1) h(-1)) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3 degrees GL) in the two-stage system.

Bioprocess and Biosystems Engineering, 2006

The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially ca... more The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially capable of giving very high ethanol productivity-was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30°C and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l À1 and ethanol concentrations of 31 and 65 g l À1 were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l À1 in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l À1 h À1 ) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3°GL) in the two-stage system.

Oil & Gas Science and Technology – Revue d’IFP Energies nouvelles, 2013

and available online here Cet article fait partie du dossier thématique ci-dessous publié dans la... more and available online here Cet article fait partie du dossier thématique ci-dessous publié dans la revue OGST, Vol. 68, n°4, pp. 621-783 et téléchargeable ici D o s s i e r DOSSIER Edited by/Sous la direction de : A. Daudin et A. Quignard

L’objectif est l’intensification de la production d’éthanol biocarburant par Saccharomyces cerevi... more L’objectif est l’intensification de la production d’éthanol biocarburant par Saccharomyces cerevisiae dans un Bioréacteur Bi-étagé avec Recyclage Cellulaire (BBRC) en mode continu. A cette fin, nous avons modélisé le fonctionnement du bioréacteur et le comportement dynamique de la levure. Le modèle développé associe les équations de bilans matière et les cinétiques microbiennes en distinguant la biomasse totale et viable en fonction des états physiologiques de la levure dans les deux étages. Le principe du BBRC est l’utilisation du potentiel de la levure à son niveau optimal ; il combine le découplage spatial croissance/production d’éthanol, l’obtention de hautes densités cellulaires et la gestion de l’activité cellulaire par le recyclage de la biomasse entre les deux étages. Des cultures ont permis la quantification du comportement de la levure dans chaque étage, en régimes transitoires et permanents, pour différentes conditions opératoires. La confrontation théorie-expérience est ...

AMB Express, 2016

Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperp... more Various enzymatic cocktails were produced from two Trichoderma reesei strains, a cellulase hyperproducer strain and a strain with β-glucosidase activity overexpression. By using various carbon sources (lactose, glucose, xylose, hemicellulosic hydrolysate) for strains growth, contrasted enzymatic activities were obtained. The enzymatic cocktails presented various levels of efficiency for the hydrolysis of cellulose Avicel into glucose, in presence of xylans, or not. These latter were also hydrolyzed with different extents according to cocktails. The most efficient cocktails (TR1 and TR3) on Avicel were richer in filter paper activity (FPU) and presented a low ratio FPU/β-glucosidase activity. Cocktails TR2 and TR5 which were produced on the higher amount of hemicellulosic hydrolysate, possess both high xylanase and β-xylosidase activities, and were the most efficient for xylans hydrolysis. When hydrolysis of Avicel was conducted in presence of xylans, a decrease of glucose release occurred for all cocktails compared to hydrolysis of Avicel alone. Mixing TR1 and TR5 cocktails with two different ratios of proteins (1/1 and 1/4) resulted in a gain of efficiency for glucose release during hydrolysis of Avicel in presence of xylans compared to TR5 alone. Our results demonstrate the importance of combining hemicellulase and cellulase activities to improve the yields of glucose release from Avicel in presence of xylans. In this context, strategies involving enzymes production with carbon sources comprising mixed C5 and C6 sugars or combining different cocktails produced on C5 or on C6 sugars are of interest for processes developed in the context of lignocellulosic biorefinery.

: Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck ... more : Enzymatic hydrolysis of lignocellulose is often considered to be the major economic bottleneck of the production process of bioethanol from lignocellulose. It is generally admitted that the most efficient organism for the production of cellulolytic enzymes is the fungus Trichoderma reesei, mostly due to its high secretion capacity. Unfortunately, this fungus secretes very low concentrations of β-glucosidase, thereby often requiring β-glucosidase supplementation for complete cellulose hydrolysis. It is especially important to have sufficient quantities of β-glucosidase in order to prevent inhibition of cellobiohydrolases by cellobiose. In order to optimize the produced cocktail, a more efficient β-glucosidase was cloned into T. reesei CL847 strain. The new strain, called CL847 TR3002, secretes the evolved β-glucosidase and was tested for cellulase production in laboratory-scale reactors. Its growth kinetics and cellulase production were characterized using fed-batch and chemostat m...

A simple kinetic model of cellulase production by Trichoderma reesei on lactose was developed and... more A simple kinetic model of cellulase production by Trichoderma reesei on lactose was developed and parameters identified using bibliographic and personal data. Assuming a mean representation of industrial hyperproducer strains, this model was used to simulate and compare several cultivation strategies regarding three criteria: in addition to usual productivity and yield calculation, oxygen demand was studied and used as a constraint for protocol design. Results showed that none of the protocols maximizes both productivity and yield, requiring to find a compromise for these parameters. Moreover, substrate concentration in the feed was the main criterion for the choice of the protocol, while oxygen demand and biomass concentration were the main issues to reach high productivities. The model will be useful for an economic study of cultivation strategies.