SCILS - Systematic consideration of inhomogeneity at the large scale: towards a stringent development of industrial bioprocesses

Lead Research Organisation: Loughborough University
Department Name: Chemical Engineering

Abstract

The proposal relates to the UK contribution to an approved ERA-IB 3rd Transnational project (within ERA-NET Scheme of the 7th EU Framework Programme) led by Professor Marco Oldiges, Institute of Bio- and Geosciences, IBG-1: Biotechnology, Forschungszentrum Jülich, FRG.

Biotechnological production in large scale bioreactors is a state-of-the-art technology. Nevertheless, full scale production is often accompanied by loss of performance compared to lab scale conditions, due to the effects of increasing bioreactor inhomogeneity. For example, cells experience a varying dissolved oxygen (DO) concentration as they are convected in the flow around a large scale bioreactor; in contrast, in small scale bioreactors it is much easier to ensure uniform DO concentrations and hence cells respire and grow in the same way throughout the fermenter. The inhomogeneities in the large scale environment, can lead to heterogeneous populations of cells, which is undesirable. Application of conventional scale-up criteria to match hydrodynamic conditions between different scales is rather difficult; the presence of inhomogeneities cannot simply be overcome at production scales by mixing more intensely, since the required energy inputs are not economically feasible. These inhomogeneity issues are not usually considered at the early stages of engineering and selecting suitable strains of organism for bioproduction, nor during lab-scale bioprocess development. Not surprisingly, this leads to critical points and failures during scale-up, necessitating additional iterations of strain or process engineering to achieve successful and economic production performance. Despite the great advances in strain engineering and understanding of cellular regulatory processes, the consideration of scale up effects such as an oscillatory environment for the biological system is mostly missing. Closing this gap can make the difference between economic efficiency and inefficiency and can provide more efficientprocesses at large scale.

Technical Summary

The project aims to tackle the challenges of production scale bioreactor inhomogeneity (e.g. of dissolved oxygen (DO) concentration) using computational and experimental procedures. Computational fluid dynamics (CFD) will be used to describe bioreactor inhomogeneity at different scales and to simulate the environmental changes experienced by individual cells. This will be accompanied by a pioneering effort to develop flow-following mobile process sensors to validate the CFD and to apply next generation laser multi-capture signal technology for online bioprocess analytics of cell morphology and physiology.

The project will focus on DO inhomogeneities as a primary target. Project partners will engineer C. glutamicum strains with reduced oxygen demand as well as improved cellular oxygen buffer capacity. A cadaverine (1,5- diaminopentane) producing C. glutamicum strain will be used as an application example; cadaverine is a potential substitute for fossil diamine monomers for polyamide polymers.

To simulate bioreactor inhomogeneity at lab scale, an experimental scale-down bioreactor will be used to characterize bioprocess performance of cadaverine producing C. glutamicum strains in the presence of DO inhomogeneities. This will be strongly supported by multi-omics investigation to elucidate changes in microbial physiology and metabolic network operation assisted by advanced stoichiometric network modelling.

The project combines all required competencies to develop next generation strains and techniques to elucidate microbial behaviour at large scale using: 1) scaled-down simulator bioreactor studies for lab scale analysis of inhomogeneities, 2) development of novel process analytical tools for next generation bioprocess characterisation, 3) engineering of microbial systems for improved biological robustness and 4) evaluation of bioreactor inhomogeneity by combining CFD with metabolic models for in-silico prediction of large scale fermentation performance.

Planned Impact

The current project proposal is aligned with several topics relevant to ERA-IB and therefore is industrially highly relevant. The change of resources from a fossil-based economy to one which significantly utilizes renewable resources for production is a global challenge. A Knowledge-Based Bioeconomy (KBBE), which is an important aspect of the European Research Framework, requires a steep increase in the realization of enhanced biotechnological processes, leading to more and more bio-based intermediate compounds to replace chemical intermediates based on fossil resources. To enable sufficient production capacity and competitive manufacturing costs for these bio-based intermediates, production will need to take place in large scale bioreactors of several hundred cubic metres. Scientists and decision makers within industrial biotechnology tell us that very large scale bioreactors will be necessary to fulfil the constraint of high capacity and low production costs. This will further increase the issues related to inhomogeneity in those bioreactors, highlighting the ultimate significance and impact of this ERA-IB proposal.

The research results and knowledge obtained from this project proposal will be relevant to all companies who are active in industrial biotechnology and manufacture using large scale fermentations. Thus, the proposal is of interest to a substantial number of European chemical and biotechnology companies, to whom the partners have contacts via previous projects already (e.g. BASF, DSM, Evonik, Sandoz, Wacker, AkzoNobel, Henkel, Novozymes). During formulation of the pre-proposal, it became apparent that it would be difficult to integrate several industrial companies, due to the limited number of total project partners allowed in the ERA-IB call, conflicting interests and internal company restrictions to share relevant production scale information. Evonik Industries AG (Halle/Westfalen, Germany), Wacker Chemie AG (Burghausen, Germany) and Antibioticos S.A. (Leon, Spain) are very much interested in the topic of the project and would like to get information about the progress of the project, documented by their letter of intent.

This project has commitment from two companies to actively participate in the project, which will be important in delivering impact. The Sequip S+E GmbH located in Dusseldorf (Germany) is a world leader in particle analysis using advanced laser multi capture signal analysis (MCSA) technology to analyse microbial cells; the operation of the laser MCSA probe is not optimized for analysis of microbial suspension, but the company's competence is not in the microbial field. The academics will work with the company to develop best-practice guidelines to utilize their technology to extract biologically and process-related information for microbial suspensions. The company will create impact by broadening the market for their process analytical technologies to meet the expected increase of products obtained by microbial fermentations.

The Vitalys company is a SME located in Denmark who have experience of strain engineering by metabolic engineering; they also operate C. glutamicum fermentations in production scale bioreactors up to 400 m3. Thus this company is directly interested in improved understanding of the effects of bioreactor inhomogeneity on cell populations and provides a link between academics and end-users of the technologies. Vitalys is interested to decrease the oxygen demand of C. glutamicum strains and hence participates in many fields of the project. The commitment of Vitalys to join the project is a clear win-win situation. The academic partners comprehensively profit from the large scale fermentation knowledge of a commercially producing company and Vitalys has direct access to knowledge and innovation relevant for large scale production obtained in the project.

Publications

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Description We have developed CFD models for two-phase flow in large scale bioreactors, with a view to gaining better understanding of the distribution of scalar quantities, such as pH, dissolved oxygen concentration and substrate concentration, which in turn affect production rates of primary and secondary metabolites, as well as the heterogeneity of the cell population. we have taken simplified flow patterns predicted by two-phase CFD and developed a network of zones model, within which a bubble population balance can be solved, along with transport equations for the major chemical species.
Exploitation Route We are working with experimentalists who are studying scaled down bioreactors which are intended to mimic the behaviour of the large scale systems that we are modelling. We also have our own PhD student working on a scaled down bioreactor and looking at lysine and cadeverine production from a modified strain of Corynebacterium glutamicum. Our work has demonstrated the relationship between the the operating characterstics of the scaled down bioreactors and the distribution of conditions at large scale. The outcome is that we better understand how to design small scale experiments, allowing more robust scale-up of large fermenters.
Sectors Agriculture, Food and Drink,Chemicals,Manufacturing, including Industrial Biotechology

 
Description SCILS 
Organisation INBIOTEC: Instituto de Biotecnología de León
Country Spain 
Sector Charity/Non Profit 
PI Contribution The project aims to systematically elucidate the influence of increasing bioreactor inhomogeneity which occurs in industrial-scale bioreactor, with respect to microbial physiology and production performance of Corynebacterium glutamicum, a microorganism with broad industrial applications. Early consideration of inhomogeneity issues during lab scale process development will facilitate the selection of the most potent production strain, accelerate the upscaling process and improve the performance at production scale. Such inhomogeneous conditions can be mimicked at lab scale by a so called scale-down simulator bioreactor, consisting of a well-mixed stirred tank reactor (STR) and a plug-flow reactor (PFR) connected in series to it. During operation the cultivation volume is continuously pumped from the STR through the PFR simulating zones of inhomogeneity known to be present at the large scale. This central challenge of inhomogeneity is addressed, for the first time, by an effort bridging both, the cellular and the process level, enabled by scale-down simulator bioreactor technology, innovative process analytical technology, multi-omics analysis and genetic engineering. Our objectives of the project: - Evaluation of bioreactor inhomogeneity by computational fluid dynamics (CFD) and linkage with metabolic network models
Collaborator Contribution Project partner objectives: - Scale-down simulator bioreactor studies for lab scale analysis of bioreactor inhomogeneity using process and multi-omics data - Development of novel tools for advanced bioprocess characterization and analytics - Engineering of microbial systems with improved robustness to bioreactor inhomogeneity
Impact Fermentation process gradient effect on C. glutamicum 1945 ?ACT3 PTUF-LDCCOPT producing cadaverine (L18) Williams Olughu (Loughborough University, Loughborough, UK) Oral presentation at the 4th BioProScale Symposium 2016, Berlin Scale-down Studies Of Corynebacterium Glutamicum Fed-Batch Cultivations To Produce Cadaverine Williams Olughu, Loughborough University, UK European Symposium on Biochemical Engineering Sciences (ESBES) 2016 11-14 September 2016, Dublin, Ireland
Start Year 2013
 
Description SCILS 
Organisation Julich Research Centre
Country Germany 
Sector Public 
PI Contribution The project aims to systematically elucidate the influence of increasing bioreactor inhomogeneity which occurs in industrial-scale bioreactor, with respect to microbial physiology and production performance of Corynebacterium glutamicum, a microorganism with broad industrial applications. Early consideration of inhomogeneity issues during lab scale process development will facilitate the selection of the most potent production strain, accelerate the upscaling process and improve the performance at production scale. Such inhomogeneous conditions can be mimicked at lab scale by a so called scale-down simulator bioreactor, consisting of a well-mixed stirred tank reactor (STR) and a plug-flow reactor (PFR) connected in series to it. During operation the cultivation volume is continuously pumped from the STR through the PFR simulating zones of inhomogeneity known to be present at the large scale. This central challenge of inhomogeneity is addressed, for the first time, by an effort bridging both, the cellular and the process level, enabled by scale-down simulator bioreactor technology, innovative process analytical technology, multi-omics analysis and genetic engineering. Our objectives of the project: - Evaluation of bioreactor inhomogeneity by computational fluid dynamics (CFD) and linkage with metabolic network models
Collaborator Contribution Project partner objectives: - Scale-down simulator bioreactor studies for lab scale analysis of bioreactor inhomogeneity using process and multi-omics data - Development of novel tools for advanced bioprocess characterization and analytics - Engineering of microbial systems with improved robustness to bioreactor inhomogeneity
Impact Fermentation process gradient effect on C. glutamicum 1945 ?ACT3 PTUF-LDCCOPT producing cadaverine (L18) Williams Olughu (Loughborough University, Loughborough, UK) Oral presentation at the 4th BioProScale Symposium 2016, Berlin Scale-down Studies Of Corynebacterium Glutamicum Fed-Batch Cultivations To Produce Cadaverine Williams Olughu, Loughborough University, UK European Symposium on Biochemical Engineering Sciences (ESBES) 2016 11-14 September 2016, Dublin, Ireland
Start Year 2013
 
Description SCILS 
Organisation SINTEF
Country Norway 
Sector Multiple 
PI Contribution The project aims to systematically elucidate the influence of increasing bioreactor inhomogeneity which occurs in industrial-scale bioreactor, with respect to microbial physiology and production performance of Corynebacterium glutamicum, a microorganism with broad industrial applications. Early consideration of inhomogeneity issues during lab scale process development will facilitate the selection of the most potent production strain, accelerate the upscaling process and improve the performance at production scale. Such inhomogeneous conditions can be mimicked at lab scale by a so called scale-down simulator bioreactor, consisting of a well-mixed stirred tank reactor (STR) and a plug-flow reactor (PFR) connected in series to it. During operation the cultivation volume is continuously pumped from the STR through the PFR simulating zones of inhomogeneity known to be present at the large scale. This central challenge of inhomogeneity is addressed, for the first time, by an effort bridging both, the cellular and the process level, enabled by scale-down simulator bioreactor technology, innovative process analytical technology, multi-omics analysis and genetic engineering. Our objectives of the project: - Evaluation of bioreactor inhomogeneity by computational fluid dynamics (CFD) and linkage with metabolic network models
Collaborator Contribution Project partner objectives: - Scale-down simulator bioreactor studies for lab scale analysis of bioreactor inhomogeneity using process and multi-omics data - Development of novel tools for advanced bioprocess characterization and analytics - Engineering of microbial systems with improved robustness to bioreactor inhomogeneity
Impact Fermentation process gradient effect on C. glutamicum 1945 ?ACT3 PTUF-LDCCOPT producing cadaverine (L18) Williams Olughu (Loughborough University, Loughborough, UK) Oral presentation at the 4th BioProScale Symposium 2016, Berlin Scale-down Studies Of Corynebacterium Glutamicum Fed-Batch Cultivations To Produce Cadaverine Williams Olughu, Loughborough University, UK European Symposium on Biochemical Engineering Sciences (ESBES) 2016 11-14 September 2016, Dublin, Ireland
Start Year 2013
 
Description SCILS 
Organisation Sequip S and E GmbH
Country Germany 
Sector Private 
PI Contribution The project aims to systematically elucidate the influence of increasing bioreactor inhomogeneity which occurs in industrial-scale bioreactor, with respect to microbial physiology and production performance of Corynebacterium glutamicum, a microorganism with broad industrial applications. Early consideration of inhomogeneity issues during lab scale process development will facilitate the selection of the most potent production strain, accelerate the upscaling process and improve the performance at production scale. Such inhomogeneous conditions can be mimicked at lab scale by a so called scale-down simulator bioreactor, consisting of a well-mixed stirred tank reactor (STR) and a plug-flow reactor (PFR) connected in series to it. During operation the cultivation volume is continuously pumped from the STR through the PFR simulating zones of inhomogeneity known to be present at the large scale. This central challenge of inhomogeneity is addressed, for the first time, by an effort bridging both, the cellular and the process level, enabled by scale-down simulator bioreactor technology, innovative process analytical technology, multi-omics analysis and genetic engineering. Our objectives of the project: - Evaluation of bioreactor inhomogeneity by computational fluid dynamics (CFD) and linkage with metabolic network models
Collaborator Contribution Project partner objectives: - Scale-down simulator bioreactor studies for lab scale analysis of bioreactor inhomogeneity using process and multi-omics data - Development of novel tools for advanced bioprocess characterization and analytics - Engineering of microbial systems with improved robustness to bioreactor inhomogeneity
Impact Fermentation process gradient effect on C. glutamicum 1945 ?ACT3 PTUF-LDCCOPT producing cadaverine (L18) Williams Olughu (Loughborough University, Loughborough, UK) Oral presentation at the 4th BioProScale Symposium 2016, Berlin Scale-down Studies Of Corynebacterium Glutamicum Fed-Batch Cultivations To Produce Cadaverine Williams Olughu, Loughborough University, UK European Symposium on Biochemical Engineering Sciences (ESBES) 2016 11-14 September 2016, Dublin, Ireland
Start Year 2013
 
Description SCILS 
Organisation Technical University Berlin
Country Germany 
Sector Academic/University 
PI Contribution The project aims to systematically elucidate the influence of increasing bioreactor inhomogeneity which occurs in industrial-scale bioreactor, with respect to microbial physiology and production performance of Corynebacterium glutamicum, a microorganism with broad industrial applications. Early consideration of inhomogeneity issues during lab scale process development will facilitate the selection of the most potent production strain, accelerate the upscaling process and improve the performance at production scale. Such inhomogeneous conditions can be mimicked at lab scale by a so called scale-down simulator bioreactor, consisting of a well-mixed stirred tank reactor (STR) and a plug-flow reactor (PFR) connected in series to it. During operation the cultivation volume is continuously pumped from the STR through the PFR simulating zones of inhomogeneity known to be present at the large scale. This central challenge of inhomogeneity is addressed, for the first time, by an effort bridging both, the cellular and the process level, enabled by scale-down simulator bioreactor technology, innovative process analytical technology, multi-omics analysis and genetic engineering. Our objectives of the project: - Evaluation of bioreactor inhomogeneity by computational fluid dynamics (CFD) and linkage with metabolic network models
Collaborator Contribution Project partner objectives: - Scale-down simulator bioreactor studies for lab scale analysis of bioreactor inhomogeneity using process and multi-omics data - Development of novel tools for advanced bioprocess characterization and analytics - Engineering of microbial systems with improved robustness to bioreactor inhomogeneity
Impact Fermentation process gradient effect on C. glutamicum 1945 ?ACT3 PTUF-LDCCOPT producing cadaverine (L18) Williams Olughu (Loughborough University, Loughborough, UK) Oral presentation at the 4th BioProScale Symposium 2016, Berlin Scale-down Studies Of Corynebacterium Glutamicum Fed-Batch Cultivations To Produce Cadaverine Williams Olughu, Loughborough University, UK European Symposium on Biochemical Engineering Sciences (ESBES) 2016 11-14 September 2016, Dublin, Ireland
Start Year 2013
 
Description SCILS 
Organisation Vitalys I/S
Country Denmark 
Sector Private 
PI Contribution The project aims to systematically elucidate the influence of increasing bioreactor inhomogeneity which occurs in industrial-scale bioreactor, with respect to microbial physiology and production performance of Corynebacterium glutamicum, a microorganism with broad industrial applications. Early consideration of inhomogeneity issues during lab scale process development will facilitate the selection of the most potent production strain, accelerate the upscaling process and improve the performance at production scale. Such inhomogeneous conditions can be mimicked at lab scale by a so called scale-down simulator bioreactor, consisting of a well-mixed stirred tank reactor (STR) and a plug-flow reactor (PFR) connected in series to it. During operation the cultivation volume is continuously pumped from the STR through the PFR simulating zones of inhomogeneity known to be present at the large scale. This central challenge of inhomogeneity is addressed, for the first time, by an effort bridging both, the cellular and the process level, enabled by scale-down simulator bioreactor technology, innovative process analytical technology, multi-omics analysis and genetic engineering. Our objectives of the project: - Evaluation of bioreactor inhomogeneity by computational fluid dynamics (CFD) and linkage with metabolic network models
Collaborator Contribution Project partner objectives: - Scale-down simulator bioreactor studies for lab scale analysis of bioreactor inhomogeneity using process and multi-omics data - Development of novel tools for advanced bioprocess characterization and analytics - Engineering of microbial systems with improved robustness to bioreactor inhomogeneity
Impact Fermentation process gradient effect on C. glutamicum 1945 ?ACT3 PTUF-LDCCOPT producing cadaverine (L18) Williams Olughu (Loughborough University, Loughborough, UK) Oral presentation at the 4th BioProScale Symposium 2016, Berlin Scale-down Studies Of Corynebacterium Glutamicum Fed-Batch Cultivations To Produce Cadaverine Williams Olughu, Loughborough University, UK European Symposium on Biochemical Engineering Sciences (ESBES) 2016 11-14 September 2016, Dublin, Ireland
Start Year 2013