Influence of fungal morphology on the performance of industrial fermentation processes for enzyme production

Daniela Alejandra Quintanilla Hernandez

Research output: Book/ReportPh.D. thesisResearch

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Abstract

Production of industrial enzymes is usually carried out as submerged aerobic fermentations. Filamentous microorganisms are widely used as hosts in these processes due to multiple advantages. Nevertheless, they also present major drawbacks, due to the unavoidable oxygen transfer limitations as a consequence of the high viscosity of the medium that they develop, which is believed to be related to the biomass concentration, growth rate and morphology. This last variable is one of the most outstanding characteristics of the filamentous fungi due to its great complexity and it was extensively studied in this work, along with its correlation to viscosity and other process variables.
Considerable research work has been conducted through the years to study fungal morphology and its relation to productivity. However, the work reported in the literature lacks relevant industrial data. In this work, a platform was developed which was able to produce high enzyme titers in comparison with what has been reported thus far in fed-batch fermentation using a soluble inducer (lactose). Different nitrogen sources were compared, and it was found that soy meal allowed for higher enzyme titers compared to what has been reported in the literature.
The developed platform was used to study the influence of agitation intensity on the morphology, rheology and protein production capability of Trichoderma reesei RUT-C30. Eight fed-batch fermentations were conducted in bench scale fermenters at two different media concentrations and four different agitation speeds. The morphology was measured with laser diffraction and the 90th percentile of the particle size distribution (PSD) was chosen as the characteristic morphology parameter. No significant difference in biomass concentration, carbon dioxide production rate and enzyme production was observed as a function of agitation speed, even at the very high power inputs. However, the morphology and rheology were considerably affected. The data produced was used to create a novel method to predict filamentous fungi rheology based on simple measurements of biomass and morphology.
Thus, morphology is an important variable in industrial submerged fermentation since it highly impacts the broth rheology. Therefore, it is important to understand the factors that affect it. One important factor is agitation-induced fragmentation since it will dictate the size of the particles, which will then affect rheology. A well-established state of the art function, the Energy Dissipation Circulation Function (EDCF), has been used to correlate hyphal fragmentation over a range of scales and impeller types. This correlation was however developed for non-growing systems (off-line fragmentation), and no attempts have been made for testing its application across different scales in actual fermentation broths. Thus, to test the validity of this correlation, a scale-down experiment was carried out. A production batch from Novozymes A/S operated in a production scale bioreactor (≈ 100 m3) was scaled down to pilot scale (≈1 m3) and to bench scale (≈0.001 m3). The EDCF was calculated for each batch along with other mixing parameters and they were correlated to the characteristic morphological parameter, the 90th percentile of the PSD. The data showed that other more simple scale up parameters are equally good at predicting mycelial fragmentation across scales, compared to the EDCF.
Furthermore, the morphological development of an industrial strain of T. reesei was monitored in pilot scale fermentations. This study showed that the morphology monitored with laser diffraction also granted the possibility to study direct physiological responses to environmental conditions in stirred bioreactors. The obtained results indicate that the nutrient depletion induced foraging due to starvation, which caused the increase in hyphal length.  
Finally, a novel, fast and easy method for statistically-verified quantification of relative hyphal tensile strength was developed in the last part of this PhD project. Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. The applicability of this novel method was demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy conditions for two strains of Aspergillus nidulans. Both strains were grown in shake flasks, and relative hyphal tensile strength was compared. The findings confirmed the utility of the developed method in strain selection and process development.
This PhD thesis brings more knowledge to the understanding of the relationship between growth kinetics, environmental conditions and the morphological structure of the filamentous fungi, which can help to tailor the morphology for a given industrial strain.
Original languageEnglish
Place of PublicationKgs. Lyngby
PublisherTechnical University of Denmark
Number of pages140
Publication statusPublished - 2017

Cite this

Quintanilla Hernandez, D. A. (2017). Influence of fungal morphology on the performance of industrial fermentation processes for enzyme production. Kgs. Lyngby: Technical University of Denmark.
Quintanilla Hernandez, Daniela Alejandra. / Influence of fungal morphology on the performance of industrial fermentation processes for enzyme production. Kgs. Lyngby : Technical University of Denmark, 2017. 140 p.
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abstract = "Production of industrial enzymes is usually carried out as submerged aerobic fermentations. Filamentous microorganisms are widely used as hosts in these processes due to multiple advantages. Nevertheless, they also present major drawbacks, due to the unavoidable oxygen transfer limitations as a consequence of the high viscosity of the medium that they develop, which is believed to be related to the biomass concentration, growth rate and morphology. This last variable is one of the most outstanding characteristics of the filamentous fungi due to its great complexity and it was extensively studied in this work, along with its correlation to viscosity and other process variables. Considerable research work has been conducted through the years to study fungal morphology and its relation to productivity. However, the work reported in the literature lacks relevant industrial data. In this work, a platform was developed which was able to produce high enzyme titers in comparison with what has been reported thus far in fed-batch fermentation using a soluble inducer (lactose). Different nitrogen sources were compared, and it was found that soy meal allowed for higher enzyme titers compared to what has been reported in the literature. The developed platform was used to study the influence of agitation intensity on the morphology, rheology and protein production capability of Trichoderma reesei RUT-C30. Eight fed-batch fermentations were conducted in bench scale fermenters at two different media concentrations and four different agitation speeds. The morphology was measured with laser diffraction and the 90th percentile of the particle size distribution (PSD) was chosen as the characteristic morphology parameter. No significant difference in biomass concentration, carbon dioxide production rate and enzyme production was observed as a function of agitation speed, even at the very high power inputs. However, the morphology and rheology were considerably affected. The data produced was used to create a novel method to predict filamentous fungi rheology based on simple measurements of biomass and morphology. Thus, morphology is an important variable in industrial submerged fermentation since it highly impacts the broth rheology. Therefore, it is important to understand the factors that affect it. One important factor is agitation-induced fragmentation since it will dictate the size of the particles, which will then affect rheology. A well-established state of the art function, the Energy Dissipation Circulation Function (EDCF), has been used to correlate hyphal fragmentation over a range of scales and impeller types. This correlation was however developed for non-growing systems (off-line fragmentation), and no attempts have been made for testing its application across different scales in actual fermentation broths. Thus, to test the validity of this correlation, a scale-down experiment was carried out. A production batch from Novozymes A/S operated in a production scale bioreactor (≈ 100 m3) was scaled down to pilot scale (≈1 m3) and to bench scale (≈0.001 m3). The EDCF was calculated for each batch along with other mixing parameters and they were correlated to the characteristic morphological parameter, the 90th percentile of the PSD. The data showed that other more simple scale up parameters are equally good at predicting mycelial fragmentation across scales, compared to the EDCF. Furthermore, the morphological development of an industrial strain of T. reesei was monitored in pilot scale fermentations. This study showed that the morphology monitored with laser diffraction also granted the possibility to study direct physiological responses to environmental conditions in stirred bioreactors. The obtained results indicate that the nutrient depletion induced foraging due to starvation, which caused the increase in hyphal length.   Finally, a novel, fast and easy method for statistically-verified quantification of relative hyphal tensile strength was developed in the last part of this PhD project. Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. The applicability of this novel method was demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy conditions for two strains of Aspergillus nidulans. Both strains were grown in shake flasks, and relative hyphal tensile strength was compared. The findings confirmed the utility of the developed method in strain selection and process development. This PhD thesis brings more knowledge to the understanding of the relationship between growth kinetics, environmental conditions and the morphological structure of the filamentous fungi, which can help to tailor the morphology for a given industrial strain.",
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Quintanilla Hernandez, DA 2017, Influence of fungal morphology on the performance of industrial fermentation processes for enzyme production. Technical University of Denmark, Kgs. Lyngby.

Influence of fungal morphology on the performance of industrial fermentation processes for enzyme production. / Quintanilla Hernandez, Daniela Alejandra.

Kgs. Lyngby : Technical University of Denmark, 2017. 140 p.

Research output: Book/ReportPh.D. thesisResearch

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AU - Quintanilla Hernandez, Daniela Alejandra

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N2 - Production of industrial enzymes is usually carried out as submerged aerobic fermentations. Filamentous microorganisms are widely used as hosts in these processes due to multiple advantages. Nevertheless, they also present major drawbacks, due to the unavoidable oxygen transfer limitations as a consequence of the high viscosity of the medium that they develop, which is believed to be related to the biomass concentration, growth rate and morphology. This last variable is one of the most outstanding characteristics of the filamentous fungi due to its great complexity and it was extensively studied in this work, along with its correlation to viscosity and other process variables. Considerable research work has been conducted through the years to study fungal morphology and its relation to productivity. However, the work reported in the literature lacks relevant industrial data. In this work, a platform was developed which was able to produce high enzyme titers in comparison with what has been reported thus far in fed-batch fermentation using a soluble inducer (lactose). Different nitrogen sources were compared, and it was found that soy meal allowed for higher enzyme titers compared to what has been reported in the literature. The developed platform was used to study the influence of agitation intensity on the morphology, rheology and protein production capability of Trichoderma reesei RUT-C30. Eight fed-batch fermentations were conducted in bench scale fermenters at two different media concentrations and four different agitation speeds. The morphology was measured with laser diffraction and the 90th percentile of the particle size distribution (PSD) was chosen as the characteristic morphology parameter. No significant difference in biomass concentration, carbon dioxide production rate and enzyme production was observed as a function of agitation speed, even at the very high power inputs. However, the morphology and rheology were considerably affected. The data produced was used to create a novel method to predict filamentous fungi rheology based on simple measurements of biomass and morphology. Thus, morphology is an important variable in industrial submerged fermentation since it highly impacts the broth rheology. Therefore, it is important to understand the factors that affect it. One important factor is agitation-induced fragmentation since it will dictate the size of the particles, which will then affect rheology. A well-established state of the art function, the Energy Dissipation Circulation Function (EDCF), has been used to correlate hyphal fragmentation over a range of scales and impeller types. This correlation was however developed for non-growing systems (off-line fragmentation), and no attempts have been made for testing its application across different scales in actual fermentation broths. Thus, to test the validity of this correlation, a scale-down experiment was carried out. A production batch from Novozymes A/S operated in a production scale bioreactor (≈ 100 m3) was scaled down to pilot scale (≈1 m3) and to bench scale (≈0.001 m3). The EDCF was calculated for each batch along with other mixing parameters and they were correlated to the characteristic morphological parameter, the 90th percentile of the PSD. The data showed that other more simple scale up parameters are equally good at predicting mycelial fragmentation across scales, compared to the EDCF. Furthermore, the morphological development of an industrial strain of T. reesei was monitored in pilot scale fermentations. This study showed that the morphology monitored with laser diffraction also granted the possibility to study direct physiological responses to environmental conditions in stirred bioreactors. The obtained results indicate that the nutrient depletion induced foraging due to starvation, which caused the increase in hyphal length.   Finally, a novel, fast and easy method for statistically-verified quantification of relative hyphal tensile strength was developed in the last part of this PhD project. Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. The applicability of this novel method was demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy conditions for two strains of Aspergillus nidulans. Both strains were grown in shake flasks, and relative hyphal tensile strength was compared. The findings confirmed the utility of the developed method in strain selection and process development. This PhD thesis brings more knowledge to the understanding of the relationship between growth kinetics, environmental conditions and the morphological structure of the filamentous fungi, which can help to tailor the morphology for a given industrial strain.

AB - Production of industrial enzymes is usually carried out as submerged aerobic fermentations. Filamentous microorganisms are widely used as hosts in these processes due to multiple advantages. Nevertheless, they also present major drawbacks, due to the unavoidable oxygen transfer limitations as a consequence of the high viscosity of the medium that they develop, which is believed to be related to the biomass concentration, growth rate and morphology. This last variable is one of the most outstanding characteristics of the filamentous fungi due to its great complexity and it was extensively studied in this work, along with its correlation to viscosity and other process variables. Considerable research work has been conducted through the years to study fungal morphology and its relation to productivity. However, the work reported in the literature lacks relevant industrial data. In this work, a platform was developed which was able to produce high enzyme titers in comparison with what has been reported thus far in fed-batch fermentation using a soluble inducer (lactose). Different nitrogen sources were compared, and it was found that soy meal allowed for higher enzyme titers compared to what has been reported in the literature. The developed platform was used to study the influence of agitation intensity on the morphology, rheology and protein production capability of Trichoderma reesei RUT-C30. Eight fed-batch fermentations were conducted in bench scale fermenters at two different media concentrations and four different agitation speeds. The morphology was measured with laser diffraction and the 90th percentile of the particle size distribution (PSD) was chosen as the characteristic morphology parameter. No significant difference in biomass concentration, carbon dioxide production rate and enzyme production was observed as a function of agitation speed, even at the very high power inputs. However, the morphology and rheology were considerably affected. The data produced was used to create a novel method to predict filamentous fungi rheology based on simple measurements of biomass and morphology. Thus, morphology is an important variable in industrial submerged fermentation since it highly impacts the broth rheology. Therefore, it is important to understand the factors that affect it. One important factor is agitation-induced fragmentation since it will dictate the size of the particles, which will then affect rheology. A well-established state of the art function, the Energy Dissipation Circulation Function (EDCF), has been used to correlate hyphal fragmentation over a range of scales and impeller types. This correlation was however developed for non-growing systems (off-line fragmentation), and no attempts have been made for testing its application across different scales in actual fermentation broths. Thus, to test the validity of this correlation, a scale-down experiment was carried out. A production batch from Novozymes A/S operated in a production scale bioreactor (≈ 100 m3) was scaled down to pilot scale (≈1 m3) and to bench scale (≈0.001 m3). The EDCF was calculated for each batch along with other mixing parameters and they were correlated to the characteristic morphological parameter, the 90th percentile of the PSD. The data showed that other more simple scale up parameters are equally good at predicting mycelial fragmentation across scales, compared to the EDCF. Furthermore, the morphological development of an industrial strain of T. reesei was monitored in pilot scale fermentations. This study showed that the morphology monitored with laser diffraction also granted the possibility to study direct physiological responses to environmental conditions in stirred bioreactors. The obtained results indicate that the nutrient depletion induced foraging due to starvation, which caused the increase in hyphal length.   Finally, a novel, fast and easy method for statistically-verified quantification of relative hyphal tensile strength was developed in the last part of this PhD project. Fungal hyphal strength is an important phenotype which can have a profound impact on bioprocess behavior. The applicability of this novel method was demonstrated by estimating relative hyphal strength during growth in control conditions and rapamycin-induced autophagy conditions for two strains of Aspergillus nidulans. Both strains were grown in shake flasks, and relative hyphal tensile strength was compared. The findings confirmed the utility of the developed method in strain selection and process development. This PhD thesis brings more knowledge to the understanding of the relationship between growth kinetics, environmental conditions and the morphological structure of the filamentous fungi, which can help to tailor the morphology for a given industrial strain.

M3 - Ph.D. thesis

BT - Influence of fungal morphology on the performance of industrial fermentation processes for enzyme production

PB - Technical University of Denmark

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ER -

Quintanilla Hernandez DA. Influence of fungal morphology on the performance of industrial fermentation processes for enzyme production. Kgs. Lyngby: Technical University of Denmark, 2017. 140 p.