Research

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15 pages, 2580 KiB

Open AccessEditor’s ChoiceArticle

Model-Based Characterization of E. coli Strains with Impaired Glucose Uptake

by Niels Krausch, Lucas Kaspersetz, Rogelio Diego Gaytán-Castro, Marie-Therese Schermeyer, Alvaro R. Lara, Guillermo Gosset, Mariano Nicolas Cruz Bournazou and Peter Neubauer

Bioengineering 2023, 10(7), 808; https://doi.org/10.3390/bioengineering10070808 - 05 Jul 2023

Cited by 2 | Viewed by 2082

Abstract

The bacterium Escherichia coli is a widely used organism in biotechnology. For high space-time yields, glucose-limited fed-batch technology is the industry standard; this is because an overflow metabolism of acetate occurs at high glucose concentrations. As an interesting alternative, various strains with limited [...] Read more.

The bacterium Escherichia coli is a widely used organism in biotechnology. For high space-time yields, glucose-limited fed-batch technology is the industry standard; this is because an overflow metabolism of acetate occurs at high glucose concentrations. As an interesting alternative, various strains with limited glucose uptake have been developed. However, these have not yet been characterized under process conditions. To demonstrate the efficiency of our previously developed high-throughput robotic platform, in the present work, we characterized three different exemplary E. coli knockout (KO) strains with limited glucose uptake capacities at three different scales (microtiter plates, 10 mL bioreactor system and 100 mL bioreactor system) under excess glucose conditions with different initial glucose concentrations. The extensive measurements of growth behavior, substrate consumption, respiration, and overflow metabolism were then used to determine the appropriate growth parameters using a mechanistic mathematical model, which allowed for a comprehensive comparative analysis of the strains. The analysis was performed coherently with these different reactor configurations and the results could be successfully transferred from one platform to another. Single and double KO mutants showed reduced specific rates for substrate uptake q_Smax and acetate production q_Apmax; meanwhile, higher glucose concentrations had adverse effects on the biomass yield coefficient Y_XSem. Additional parameters compared to previous studies for the oxygen uptake rate and carbon dioxide production rate indicated differences in the specific oxygen uptake rate q_Omax. This study is an example of how automated robotic equipment, together with mathematical model-based approaches, can be successfully used to characterize strains and obtain comprehensive information more quickly, with a trade-off between throughput and analytical capacity. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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44 pages, 29600 KiB

Open AccessArticle

Holistic Approach to Process Design and Scale-Up for Itaconic Acid Production from Crude Substrates

by Katharina Maria Saur, Robert Kiefel, Paul-Joachim Niehoff, Jordy Hofstede, Philipp Ernst, Johannes Brockkötter, Jochem Gätgens, Jörn Viell, Stephan Noack, Nick Wierckx, Jochen Büchs and Andreas Jupke

Bioengineering 2023, 10(6), 723; https://doi.org/10.3390/bioengineering10060723 - 14 Jun 2023

Cited by 4 | Viewed by 2191

Abstract

Bio-based bulk chemicals such as carboxylic acids continue to struggle to compete with their fossil counterparts on an economic basis. One possibility to improve the economic feasibility is the use of crude substrates in biorefineries. However, impurities in these substrates pose challenges in [...] Read more.

Bio-based bulk chemicals such as carboxylic acids continue to struggle to compete with their fossil counterparts on an economic basis. One possibility to improve the economic feasibility is the use of crude substrates in biorefineries. However, impurities in these substrates pose challenges in fermentation and purification, requiring interdisciplinary research. This work demonstrates a holistic approach to biorefinery process development, using itaconic acid production on thick juice based on sugar beets with Ustilago sp. as an example. A conceptual process design with data from artificially prepared solutions and literature data from fermentation on glucose guides the simultaneous development of the upstream and downstream processes up to a 100 L scale. Techno-economic analysis reveals substrate consumption as the main constituent of production costs and therefore, the product yield is the driver of process economics. Aligning pH-adjusting agents in the fermentation and the downstream process is a central lever for product recovery. Experiments show that fermentation can be transferred from glucose to thick juice by changing the feeding profile. In downstream processing, an additional decolorization step is necessary to remove impurities accompanying the crude substrate. Moreover, we observe an increased use of pH-adjusting agents compared to process simulations. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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22 pages, 2370 KiB

Open AccessArticle

Downscaling Industrial-Scale Syngas Fermentation to Simulate Frequent and Irregular Dissolved Gas Concentration Shocks

by Lars Puiman, Eduardo Almeida Benalcázar, Cristian Picioreanu, Henk J. Noorman and Cees Haringa

Bioengineering 2023, 10(5), 518; https://doi.org/10.3390/bioengineering10050518 - 25 Apr 2023

Cited by 4 | Viewed by 3647

Abstract

In large-scale syngas fermentation, strong gradients in dissolved gas (CO, H₂) concentrations are very likely to occur due to locally varying mass transfer and convection rates. Using Euler-Lagrangian CFD simulations, we analyzed these gradients in an industrial-scale external-loop gas-lift reactor (EL-GLR) [...] Read more.

In large-scale syngas fermentation, strong gradients in dissolved gas (CO, H₂) concentrations are very likely to occur due to locally varying mass transfer and convection rates. Using Euler-Lagrangian CFD simulations, we analyzed these gradients in an industrial-scale external-loop gas-lift reactor (EL-GLR) for a wide range of biomass concentrations, considering CO inhibition for both CO and H₂ uptake. Lifeline analyses showed that micro-organisms are likely to experience frequent (5 to 30 s) oscillations in dissolved gas concentrations with one order of magnitude. From the lifeline analyses, we developed a conceptual scale-down simulator (stirred-tank reactor with varying stirrer speed) to replicate industrial-scale environmental fluctuations at bench scale. The configuration of the scale-down simulator can be adjusted to match a broad range of environmental fluctuations. Our results suggest a preference for industrial operation at high biomass concentrations, as this would strongly reduce inhibitory effects, provide operational flexibility and enhance the product yield. The peaks in dissolved gas concentration were hypothesized to increase the syngas-to-ethanol yield due to the fast uptake mechanisms in C. autoethanogenum. The proposed scale-down simulator can be used to validate such results and to obtain data for parametrizing lumped kinetic metabolic models that describe such short-term responses. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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14 pages, 2632 KiB

Open AccessArticle

Optimization of the Fermentative Production of Rhizomucor miehei Lipase in Aspergillus oryzae by Controlling Morphology

by Chao Li, Dou Xu, Zhiyue Xiong, Yiming Yang, Guiwei Tian, Xuezhi Wu, Yonghong Wang, Yingping Zhuang, Ju Chu and Xiwei Tian

Bioengineering 2022, 9(11), 610; https://doi.org/10.3390/bioengineering9110610 - 25 Oct 2022

Cited by 1 | Viewed by 1749

Abstract

Morphology plays an important role in the fermentation bioprocess of filamentous fungi. In this study, we investigated the controlling strategies of morphology that improved the efficiency of Rhizomucor miehei lipase (RML) production using a high-yield Aspergillus oryzae. First, the inoculated spore concentrations were [...] Read more.

Morphology plays an important role in the fermentation bioprocess of filamentous fungi. In this study, we investigated the controlling strategies of morphology that improved the efficiency of Rhizomucor miehei lipase (RML) production using a high-yield Aspergillus oryzae. First, the inoculated spore concentrations were optimized in seed culture, and the RML activity increased by 43.4% with the well-controlled mycelium pellets in both ideal sizes and concentrations. Then, the initial nitrogen source and agitation strategies were optimized to regulate the morphology of Aspergillus oryzae in a 5 L bioreactor, and the established stable fermentation system increased the RML activity to 232.0 U/mL, combined with an increase in total RML activity from 98,080 U to 487,179 U. Furthermore, the optimized fermentation strategy was verified by a high-yield Aspergillus oryzae and achieved an additional improvement of RML activity, up to 320.0 U/mL. Moreover, this optimized fermentation bioprocess was successfully scaled up to a 50 L bioreactor, and the RML activity reached 550.0 U/mL. This work has established a stable precision fermentation bioprocess for RML production by A. oryzae in bioreactors, and the controlling strategy developed in this study could potentially be extended to an industrial scale for RML production with high efficiency. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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15 pages, 3527 KiB

Open AccessArticle

Large-Scale Expansion of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Stirred Suspension Bioreactor Enabled by Computational Fluid Dynamics Modeling

by Junhong Zhang, Yan Peng, Meijin Guo and Chao Li

Bioengineering 2022, 9(7), 274; https://doi.org/10.3390/bioengineering9070274 - 23 Jun 2022

Cited by 3 | Viewed by 2427

Abstract

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) hold great potential to generate novel and curative cell therapy products. However, the current large-scale cultivation of hUCMSCs is based on empirical geometry-dependent methods, limiting the generation of high-quantity and high-quality hUCMSCs for clinical therapy. Herein, [...] Read more.

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) hold great potential to generate novel and curative cell therapy products. However, the current large-scale cultivation of hUCMSCs is based on empirical geometry-dependent methods, limiting the generation of high-quantity and high-quality hUCMSCs for clinical therapy. Herein, we develop a novel scale-up strategy based on computational fluid dynamics (CFD) to effectively expand the hUCMSCs in a 3D tank bioreactor. Using a standardized hUCMSCs line on microcarriers, we successfully translated and expanded the hUCMSCs from a 200 mL spinner flask to a 1.5 L computer-controlled bioreactor by matching the shear environment and suspending the microcarrier. Experimental results revealed that the batch-cultured hUCMSCs in bioreactors with an agitation speed of 40 rpm shared a more favorable growth and physiological state, similar to that run at 45 rpm in a 200 mL spinner flask, showing comparability in both culture systems. Notably, the maximum cell density reached up to 27.3 × 10⁵ cells/mL in fed-batch culture, 2.9 folds of that of batch culture and 20.2 times of seeding cells. As such, efficient process optimization and scale-up expansion of hUCMSCs were achieved in the microcarrier-based bioreactor system by the developed CFD simulation strategy, which provided an alternative toolbox to generate massive and standardized curative cell therapy products. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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Review

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23 pages, 3130 KiB

Open AccessReview

Bioprocessing Considerations towards the Manufacturing of Therapeutic Skeletal and Smooth Muscle Cells

by Teresa Franchi-Mendes, Marília Silva, Ana Luísa Cartaxo, Ana Fernandes-Platzgummer, Joaquim M. S. Cabral and Cláudia L. da Silva

Bioengineering 2023, 10(9), 1067; https://doi.org/10.3390/bioengineering10091067 - 09 Sep 2023

Viewed by 1343

Abstract

Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth muscle, responsible for involuntary and voluntary contraction, respectively. In this review, [...] Read more.

Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth muscle, responsible for involuntary and voluntary contraction, respectively. In this review, several features and parameters involved in the bioprocessing of muscle cells are addressed. The cell isolation process is depicted, depending on the type of tissue (smooth or skeletal muscle), followed by the description of the challenges involving the use of adult donor tissue and the strategies to overcome the hurdles of reaching relevant cell numbers towards a clinical application. Specifically, the use of stem/progenitor cells is highlighted as a source for smooth and skeletal muscle cells towards the development of a cellular product able to maintain the target cell’s identity and functionality. Moreover, taking into account the need for a robust and cost-effective bioprocess for cell manufacturing, the combination of muscle cells with biomaterials and the need for scale-up envisioning clinical applications are also approached. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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23 pages, 2506 KiB

Open AccessReview

From Spatial-Temporal Multiscale Modeling to Application: Bridging the Valley of Death in Industrial Biotechnology

by Xueting Wang, Ali Mohsin, Yifei Sun, Chao Li, Yingping Zhuang and Guan Wang

Bioengineering 2023, 10(6), 744; https://doi.org/10.3390/bioengineering10060744 - 20 Jun 2023

Viewed by 1776

Abstract

The Valley of Death confronts industrial biotechnology with a significant challenge to the commercialization of products. Fortunately, with the integration of computation, automation and artificial intelligence (AI) technology, the industrial biotechnology accelerates to cross the Valley of Death. The Fourth Industrial Revolution (Industry [...] Read more.

The Valley of Death confronts industrial biotechnology with a significant challenge to the commercialization of products. Fortunately, with the integration of computation, automation and artificial intelligence (AI) technology, the industrial biotechnology accelerates to cross the Valley of Death. The Fourth Industrial Revolution (Industry 4.0) has spurred advanced development of intelligent biomanufacturing, which has evolved the industrial structures in line with the worldwide trend. To achieve this, intelligent biomanufacturing can be structured into three main parts that comprise digitalization, modeling and intellectualization, with modeling forming a crucial link between the other two components. This paper provides an overview of mechanistic models, data-driven models and their applications in bioprocess development. We provide a detailed elaboration of the hybrid model and its applications in bioprocess engineering, including strain design, process control and optimization, as well as bioreactor scale-up. Finally, the challenges and opportunities of biomanufacturing towards Industry 4.0 are also discussed. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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17 pages, 491 KiB

Open AccessReview

rAAV Manufacturing: The Challenges of Soft Sensing during Upstream Processing

by Cristovão Freitas Iglesias, Jr., Milica Ristovski, Miodrag Bolic and Miroslava Cuperlovic-Culf

Bioengineering 2023, 10(2), 229; https://doi.org/10.3390/bioengineering10020229 - 08 Feb 2023

Cited by 5 | Viewed by 2943

Abstract

Recombinant adeno-associated virus (rAAV) is the most effective viral vector technology for directly translating the genomic revolution into medicinal therapies. However, the manufacturing of rAAV viral vectors remains challenging in the upstream processing with low rAAV yield in large-scale production and high cost, [...] Read more.

Recombinant adeno-associated virus (rAAV) is the most effective viral vector technology for directly translating the genomic revolution into medicinal therapies. However, the manufacturing of rAAV viral vectors remains challenging in the upstream processing with low rAAV yield in large-scale production and high cost, limiting the generalization of rAAV-based treatments. This situation can be improved by real-time monitoring of critical process parameters (CPP) that affect critical quality attributes (CQA). To achieve this aim, soft sensing combined with predictive modeling is an important strategy that can be used for optimizing the upstream process of rAAV production by monitoring critical process variables in real time. However, the development of soft sensors for rAAV production as a fast and low-cost monitoring approach is not an easy task. This review article describes four challenges and critically discusses the possible solutions that can enable the application of soft sensors for rAAV production monitoring. The challenges from a data scientist’s perspective are (i) a predictor variable (soft-sensor inputs) set without AAV viral titer, (ii) multi-step forecasting, (iii) multiple process phases, and (iv) soft-sensor development composed of the mechanistic model. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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23 pages, 1558 KiB

Open AccessReview

Bioprocess Economic Modeling: Decision Support Tools for the Development of Stem Cell Therapy Products

by William O. S. Salvador, Inês A. B. Ribeiro, Diogo E. S. Nogueira, Frederico C. Ferreira, Joaquim M. S. Cabral and Carlos A. V. Rodrigues

Bioengineering 2022, 9(12), 791; https://doi.org/10.3390/bioengineering9120791 - 11 Dec 2022

Cited by 2 | Viewed by 2901

Abstract

Over recent years, the field of cell and gene therapy has witnessed rapid growth due to the demonstrated benefits of using living cells as therapeutic agents in a broad range of clinical studies and trials. Bioprocess economic models (BEMs) are fundamental tools for [...] Read more.

Over recent years, the field of cell and gene therapy has witnessed rapid growth due to the demonstrated benefits of using living cells as therapeutic agents in a broad range of clinical studies and trials. Bioprocess economic models (BEMs) are fundamental tools for guiding decision-making in bioprocess design, being capable of supporting process optimization and helping to reduce production costs. These tools are particularly important when it comes to guiding manufacturing decisions and increasing the likelihood of market acceptance of cell-based therapies, which are often cost-prohibitive because of high resource and quality control costs. Not only this, but the inherent biological variability of their underlying bioprocesses makes them particularly susceptible to unforeseen costs arising from failed or delayed production batches. The present work reviews important concepts concerning the development of bioprocesses for stem cell therapy products and highlights the valuable role which BEMs can play in this endeavor. Additionally, some theoretical concepts relevant to the building and structuring of BEMs are explored. Finally, a comprehensive review of the existent BEMs so far reported in the scientific literature for stem cell-related bioprocesses is provided to showcase their potential usefulness. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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18 pages, 11050 KiB

Open AccessReview

Optimization and Scale-Up of Fermentation Processes Driven by Models

by Yuan-Hang Du, Min-Yu Wang, Lin-Hui Yang, Ling-Ling Tong, Dong-Sheng Guo and Xiao-Jun Ji

Bioengineering 2022, 9(9), 473; https://doi.org/10.3390/bioengineering9090473 - 14 Sep 2022

Cited by 19 | Viewed by 11963

Abstract

In the era of sustainable development, the use of cell factories to produce various compounds by fermentation has attracted extensive attention; however, industrial fermentation requires not only efficient production strains, but also suitable extracellular conditions and medium components, as well as scaling-up. In [...] Read more.

In the era of sustainable development, the use of cell factories to produce various compounds by fermentation has attracted extensive attention; however, industrial fermentation requires not only efficient production strains, but also suitable extracellular conditions and medium components, as well as scaling-up. In this regard, the use of biological models has received much attention, and this review will provide guidance for the rapid selection of biological models. This paper first introduces two mechanistic modeling methods, kinetic modeling and constraint-based modeling (CBM), and generalizes their applications in practice. Next, we review data-driven modeling based on machine learning (ML), and highlight the application scope of different learning algorithms. The combined use of ML and CBM for constructing hybrid models is further discussed. At the end, we also discuss the recent strategies for predicting bioreactor scale-up and culture behavior through a combination of biological models and computational fluid dynamics (CFD) models. Full article

(This article belongs to the Special Issue Design, Optimization and Scale-Up of Industrial Bioprocess)

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