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

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 efficient processes at large scale.

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.

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.