Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations
Abstract
:1. Introduction
2. Materials and Methods
2.1. HTBD Facility
2.2. Cultivation
2.3. Sampling and Analytic
2.4. Strains
2.5. Computational Methods
2.5.1. Parameter Estimation
2.5.2. Monte Carlo Simulation
2.5.3. Feed Calculation
3. Results
3.1. Parallel Cultivation
3.2. Prediction of Batch and Feed Start
3.3. Feed and Fed-Batch
3.4. Parameter Estimation
4. Discussion
5. Conclusions
- Design a specific strategy for each different clone of the conditional screening experiment.
- Increase the robustness of the robotic operation against experimental disturbance.
- Give an approximation of the reliability of the simulation results with respect to production scale performance.
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
- Dusny, C.; Grünberger, A. Microfluidic single-cell analysis in biotechnology: From monitoring towards understanding. Curr. Opin. Biotechnol. 2020, 63, 26–33. [Google Scholar] [CrossRef] [PubMed]
- Leavell, M.D.; Singh, A.H.; Kaufmann-Malaga, B.B. High-throughput screening for improved microbial cell factories, perspective and promise. Curr. Opin. Biotechnol. 2020, 62, 22–28. [Google Scholar] [CrossRef] [PubMed]
- Bunzel, H.A.; Pi, X.G.; Pott, M.; Hilvert, D. Speeding up enzyme discovery and engineering with ultrahigh-throughput methods. Curr. Opin. Struct. Biol. 2018, 48, 149–156. [Google Scholar] [CrossRef] [PubMed]
- Nicolaou, S.A.; Gaida, S.M.; Papoutsakis, E.T. A comparative view of metabolite and substrate stress and tolerance in microbial bioprocessing: From biofuels and chemicals, to biocatalysis and bioremediation. Metab. Eng. 2010, 12, 307–331. [Google Scholar] [CrossRef]
- Crater, J.S.; Lievense, J.C. Scale-up of industrial microbial processes. FEMS Microbiol. Lett. 2018, 365, 1–5. [Google Scholar] [CrossRef]
- Delvigne, F.; Takors, R.; Mudde, R.; Van Gulik, W.; Noorman, H. Bioprocess scale-up/down as integrative enabling technology: From fluid mechanics to systems biology and beyond. Microb. Biotechnol. 2017, 10, 1267–1274. [Google Scholar] [CrossRef]
- Ho, P.; Westerwalbesloh, C.; Kaganovitch, E.; Grünberger, A.; Neubauer, P.; Kohlheyer, D.; Von Lieres, E. Reproduction of Large-Scale Bioreactor Conditions on Microfluidic Chips. Microorganisms 2019, 7, 105. [Google Scholar] [CrossRef] [Green Version]
- Anane, E.; García, Á.C.; Haby, B.; Hans, S.; Krausch, N.; Krewinkel, M.; Hauptmann, P.; Neubauer, P.; Bournazou, M.N.C. A model-based framework for parallel scale-down fed-batch cultivations in mini-bioreactors for accelerated phenotyping. Biotechnol. Bioeng. 2019, 116, 2906–2918. [Google Scholar] [CrossRef] [Green Version]
- Tajsoleiman, T.; Mears, L.; Krühne, U.; Gernaey, K.V.; Cornelissen, S. An Industrial Perspective on Scale-Down Challenges Using Miniaturized Bioreactors. Trends Biotechnol. 2019, 37, 697–706. [Google Scholar] [CrossRef]
- Hemmerich, J.; Noack, S.; Wiechert, W.; Oldiges, M. Microbioreactor Systems for Accelerated Bioprocess Development. Biotechnol. J. 2018, 13, 1–25. [Google Scholar] [CrossRef]
- Janzen, N.H.; Striedner, G.; Jarmer, J.; Voigtmann, M.; Abad, S.; Reinisch, D. Implementation of a Fully Automated Microbial Cultivation Platform for Strain and Process Screening. Biotechnol. J. 2019, 14, 1800625. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kusterer, A.; Krause, C.; Kaufmann, K.; Arnold, M.; Weuster-Botz, D. Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations. Bioprocess Biosyst. Eng. 2008, 31, 207–215. [Google Scholar] [CrossRef] [PubMed]
- Morschett, H.; Freier, L.; Rohde, J.; Wiechert, W.; Von Lieres, E.; Oldiges, M. A framework for accelerated phototrophic bioprocess development: Integration of parallelized microscale cultivation, laboratory automation and Kriging-assisted experimental design. Biotechnol. Biofuels 2017, 10, 26. [Google Scholar] [CrossRef] [PubMed]
- Bareither, R.; Pollard, D. A review of advanced small-scale parallel bioreactor technology for accelerated process development: Current state and future need. Biotechnol. Prog. 2010, 27, 2–14. [Google Scholar] [CrossRef]
- Tai, M.; Ly, A.; Leung, I.; Nayar, G. Efficient high-throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system. Biotechnol. Prog. 2015, 31, 1388–1395. [Google Scholar] [CrossRef] [Green Version]
- Knorr, B.; Schlieker, H.; Hohmann, H.-P.; Weuster-Botz, D. Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis. Biochem. Eng. J. 2007, 33, 263–274. [Google Scholar] [CrossRef] [Green Version]
- Haby, B.; Hans, S.; Anane, E.; Sawatzki, A.; Krausch, N.; Neubauer, P.; Bournazou, M.N.C. Integrated Robotic Mini Bioreactor Platform for Automated, Parallel Microbial Cultivation with Online Data Handling and Process Control. SLAS Technol. Transl. Life Sci. Innov. 2019, 24, 569–582. [Google Scholar] [CrossRef]
- Newton, J.; Oeggl, R.; Janzen, N.H.; Abad, S.; Reinisch, D. Process adapted calibration improves fluorometric pH sensor precision in sophisticated fermentation processes. Eng. Life Sci. 2020, 20, 331–337. [Google Scholar] [CrossRef]
- Hemmerich, J.; Tenhaef, N.; Steffens, C.; Kappelmann, J.; Weiske, M.; Reich, S.J.; Wiechert, W.; Oldiges, M.; Noack, S. Less Sacrifice, More Insight: Repeated Low-Volume Sampling of Microbioreactor Cultivations Enables Accelerated Deep Phenotyping of Microbial Strain Libraries. Biotechnol. J. 2018, 14, 1–10. [Google Scholar] [CrossRef]
- Bournazou, M.C.; Barz, T.; Nickel, D.; Cárdenas, D.L.; Glauche, F.; Knepper, A.; Neubauer, P. Online optimal experimental re-design in robotic parallel fed-batch cultivation facilities. Biotechnol. Bioeng. 2017, 114, 610–619. [Google Scholar] [CrossRef]
- Von Stosch, M.; Willis, M.J. Intensified design of experiments for upstream bioreactors. Eng. Life Sci. 2016, 17, 1173–1184. [Google Scholar] [CrossRef] [PubMed]
- Bayer, B.; Von Stosch, M.; Striedner, G.; Duerkop, M. Comparison of Modeling Methods for DoE-Based Holistic Upstream Process Characterization. Biotechnol. J. 2020, 15, e1900551. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Hans, S.; Ulmer, C.; Narayanan, H.; Brautaset, T.; Krausch, N.; Neubauer, P.; Schäffl, I.; Sokolov, M.; Bournazou, M.N.C. Monitoring Parallel Robotic Cultivations with Online Multivariate Analysis. Processes 2020, 8, 582. [Google Scholar] [CrossRef]
- Delvigne, F.; Goffin, P. Microbial heterogeneity affects bioprocess robustness: Dynamic single-cell analysis contributes to understanding of microbial populations. Biotechnol. J. 2013, 9, 61–72. [Google Scholar] [CrossRef] [PubMed]
- Anane, E.; López C, D.C.; Neubauer, P.; Bournazou, M.N.C. Modelling overflow metabolism in Escherichia coli by acetate cycling. Biochem. Eng. J. 2017, 125, 23–30. [Google Scholar] [CrossRef] [Green Version]
- Sauer, U.; Lasko, D.R.; Fiaux, J.; Hochuli, M.; Glaser, R.; Szyperski, T.; Wüthrich, K.; Bailey, J.E. Metabolic Flux Ratio Analysis of Genetic and Environmental Modulations of Escherichia coli Central Carbon Metabolism. J. Bacteriol. 1999, 181, 6679–6688. [Google Scholar] [CrossRef] [Green Version]
- Baba, T.; Ara, T.; Hasegawa, M.; Takai, Y.; Okumura, Y.; Baba, M.; A Datsenko, K.; Tomita, M.; Wanner, B.L.; Mori, H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection. Mol. Syst. Biol. 2006, 2, 2006.0008. [Google Scholar] [CrossRef] [Green Version]
- López C, D.C.; Barz, T.; Peñuela, M.; Villegas, A.; Ochoa, S.; Wozny, G. Model-based identifiable parameter determination applied to a simultaneous saccharification and fermentation process model for bio-ethanol production. Biotechnol. Prog. 2013, 29, 1064–1082. [Google Scholar] [CrossRef]
- Hindmarsh, A.C.; Brown, P.N.; Grant, K.E.; Lee, S.L.; Serban, R.; Shumaker, D.E.; Woodward, C.S. SUNDIALS. ACM Trans. Math. Softw. 2005, 31, 363–396. [Google Scholar] [CrossRef]
- Moser, A.; Moser, P.D.A. Haggstrom, Bioprocess Technology: Fundamentals and Applications; Springer Science & Business Media: New York, NY, USA, 1988. [Google Scholar] [CrossRef]
- Krausch, N.; Barz, T.; Sawatzki, A.; Gruber, M.; Kamel, S.; Neubauer, P.; Bournazou, M.N.C. Monte Carlo Simulations for the Analysis of Non-linear Parameter Confidence Intervals in Optimal Experimental Design. Front. Bioeng. Biotechnol. 2019, 7, 122. [Google Scholar] [CrossRef]
- Barz, T.; Sommer, A.; Wilms, T.; Neubauer, P.; Bournazou, M.N.C. Adaptive optimal operation of a parallel robotic liquid handling station. IFAC-PapersOnLine 2018, 51, 765–770. [Google Scholar] [CrossRef]
- Franceschini, G.; Macchietto, S. Model-based design of experiments for parameter precision: State of the art. Chem. Eng. Sci. 2008, 63, 4846–4872. [Google Scholar] [CrossRef]
- Anane, E.; López C, D.C.; Barz, T.; Sin, G.; Gernaey, K.V.; Neubauer, P.; Bournazou, M.N.C. Output uncertainty of dynamic growth models: Effect of uncertain parameter estimates on model reliability. Biochem. Eng. J. 2019, 150, 107247. [Google Scholar] [CrossRef]
- Dynamics of Mathematical Models in Biology. Dyn. Math. Models Biol. 2016, 31–41. [CrossRef] [Green Version]
- Villaverde, A.F.; Banga, J.R. Structural Properties of Dynamic Systems Biology Models: Identifiability, Reachability, and Initial Conditions. Processes 2017, 5, 29. [Google Scholar] [CrossRef] [Green Version]
- Neubauer, P.; Lin, H.Y.; Mathiszik, B. Metabolic load of recombinant protein production: Inhibition of cellular capacities for glucose uptake and respiration after induction of a heterologous gene in Escherichia coli. Biotechnol. Bioeng. 2003, 83, 53–64. [Google Scholar] [CrossRef]
- Jabarivelisdeh, B.; Carius, L.; Findeisen, R.; Waldherr, S. Adaptive predictive control of bioprocesses with constraint-based modeling and estimation. Comput. Chem. Eng. 2020, 135, 106744. [Google Scholar] [CrossRef]
Sequential Task (Iteration) | Cultivation Time (h) | Available Measurements | |||
---|---|---|---|---|---|
DOT | Biomass | Glucose | Acetate | ||
1 | 1.38 | 321 | 6 | 0 | 0 |
2 | 1.88 | 411 | 16 | 0 | 0 |
3 | 2.55 | 531 | 16 | 10 | 10 |
4 | 3.52 | 705 | 26 | 10 | 10 |
5 | 3.93 | 780 | 26 | 20 | 20 |
6 | 5.17 | 999 | 36 | 20 | 20 |
7 | 5.94 | 1137 | 36 | 30 | 30 |
8 | 6.91 | 1311 | 46 | 30 | 30 |
9 | 7.66 | 1440 | 46 | 40 | 40 |
Strain | Glucose Consumption (hh:mm) | Acetate Consumption (hh:mm) | Feed Start (hh:mm) | ||||
---|---|---|---|---|---|---|---|
Initial | Adjusted | Observed | Initial | Adjusted | Observed | ||
E. coli W3110 | 01:46 | 01:40 | 01:39 ± 00:01 | 02:03 | 01:48 | 01:48 | 01:55 |
E. coli BW25113 | 01:52 | 01:38 | 01:49 ± 00:03 | 02:00 | 03:02 | >02:23 | 02:23 |
E. coli BW25113 ΔompT | 01:46 | 01:40 | 01:40 ± 00:01 | 01:53 | 03:05 | 02:10 | 02:23 |
E. coli BW25113 ΔaceA | 02:13 | 02:11 | 01:48 ± 00:21 | 02:22 | 03:06 | >02:37 | 02:37 |
E. coli BW25113 ΔfliA | 01:51 | 01:36 | 01:42 ± 00:01 | 01:59 | 02:13 | 02:07 | 02:16 |
E. coli BW25113 ΔgatC | 01:49 | 01:39 | 01:46 ± 00:05 | 01:57 | 02:50 | 02:25 | 02:30 |
E. coli BW25113 ΔgatZ | 01:46 | 01:43 | 01:43 ± 00:01 | 01:55 | 03:03 | >02:09 | 02:09 |
E. coli BW25113 ΔglcB | 01:55 | 01:56 | 01:51 ± 00:03 | 02:03 | 02:24 | 02:30 | 02:37 |
E.coli W3110 | E. coli BW215113 | |||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Parameter | Unit | Inital Guess | WT | ΔompT | ΔaceA | ΔfliA | ΔgatC | ΔgatZ | ΔglcB | |||||||||||||||||
θ | σθ | %σθ | θ | σθ | %σθ | θ | σθ | %σθ | θ | σθ | %σθ | θ | σθ | %σθ | θ | σθ | %σθ | θ | σθ | %σθ | θ | σθ | %σθ | |||
qAmax | g g−1 h−1 | 1.0252 | 1.59 | 0.11 | 6.78 | 0.52 | 0.11 | 21.74 | 0.90 | 0.05 | 5.92 | 0.56 | 0.15 | 26.05 | 0.72 | 0.11 | 15.59 | 0.68 | 0.08 | 12.19 | 0.86 | 0.10 | 11.54 | 0.71 | 0.07 | 9.54 |
Kaq | g L−1 | 0.2133 | 0.59 | 0.14 | 23.11 | 0.55 | 0.09 | 16.28 | 0.98 | 0.07 | 7.11 | 0.98 | 0.14 | 13.82 | 0.60 | 0.12 | 20.70 | 0.68 | 0.10 | 14.16 | 0.75 | 0.12 | 15.97 | 0.70 | 0.04 | 6.24 |
Ksq | g L−1 | 1.0667 | 1.52 | 0.33 | 21.71 | 1.98 | 0.34 | 16.98 | 1.97 | 0.26 | 13.17 | 1.91 | 0.32 | 16.95 | 1.77 | 0.29 | 16.26 | 1.99 | 0.25 | 12.35 | 1.63 | 0.31 | 18.80 | 1.68 | 0.34 | 20.14 |
Yam | g g−1 | 0.1955 | 0.40 | 0.03 | 7.18 | 0.41 | 0.04 | 10.79 | 0.44 | 0.05 | 10.16 | 0.44 | 0.08 | 18.79 | 0.48 | 0.04 | 8.04 | 0.44 | 0.08 | 17.80 | 0.44 | 0.04 | 8.89 | 0.42 | 0.03 | 6.82 |
Yaresp | g g−1 | 0.1672 | 0.15 | 0.01 | 4.94 | 0.15 | 0.01 | 5.09 | 0.15 | 0.01 | 4.55 | 0.12 | 0.01 | 8.36 | 0.15 | 0.01 | 4.79 | 0.15 | 0.01 | 8.02 | 0.13 | 0.01 | 6.65 | 0.13 | 0.00 | 3.44 |
Yem | g g−1 | 0.56 | 0.60 | 0.01 | 2.48 | 0.60 | 0.01 | 1.82 | 0.60 | 0.02 | 2.89 | 0.58 | 0.02 | 2.70 | 0.60 | 0.01 | 1.94 | 0.60 | 0.02 | 3.27 | 0.58 | 0.01 | 2.19 | 0.59 | 0.01 | 1.53 |
qSmax | g g−1 h−1 | 1.3431 | 1.60 | 0.02 | 1.20 | 1.58 | 0.03 | 2.09 | 1.60 | 0.03 | 2.02 | 1.47 | 0.03 | 1.79 | 1.59 | 0.03 | 1.83 | 1.55 | 0.03 | 2.08 | 1.39 | 0.02 | 1.16 | 1.40 | 0.04 | 2.54 |
Ks | g L−1 | 0.05 | 0.03 | 0.01 | 22.73 | 0.03 | 0.00 | 15.72 | 0.03 | 0.01 | 27.93 | 0.08 | 0.01 | 8.44 | 0.03 | 0.01 | 19.59 | 0.03 | 0.01 | 21.88 | 0.04 | 0.01 | 29.35 | 0.03 | 0.01 | 18.79 |
Ko | g L−1 | 1 | 19.87 | 1.52 | 7.64 | 18.13 | 1.87 | 10.32 | 14.51 | 1.27 | 8.76 | 18.64 | 1.93 | 10.38 | 16.29 | 1.66 | 10.17 | 16.00 | 2.22 | 13.88 | 14.13 | 0.95 | 6.69 | 9.57 | 1.27 | 13.28 |
Yosresp | g g−1 | 1 | 2.00 | 0.05 | 2.54 | 2.00 | 0.04 | 1.90 | 1.99 | 0.09 | 4.46 | 1.80 | 0.09 | 4.78 | 1.76 | 0.05 | 3.03 | 1.97 | 0.08 | 4.31 | 1.89 | 0.06 | 3.04 | 1.19 | 0.02 | 2.03 |
pAmax | g g−1 h−1 | 1.3091 | 1.60 | 0.07 | 4.18 | 0.93 | 0.12 | 13.29 | 1.13 | 0.08 | 7.31 | 0.86 | 0.07 | 8.64 | 0.95 | 0.07 | 7.45 | 0.98 | 0.09 | 8.96 | 1.08 | 0.09 | 8.37 | 0.87 | 0.09 | 10.77 |
Yaof | g g−1 | 0.4607 | 0.35 | 0.01 | 3.88 | 0.20 | 0.02 | 12.23 | 0.24 | 0.02 | 7.52 | 0.21 | 0.02 | 6.99 | 0.21 | 0.02 | 7.05 | 0.20 | 0.02 | 8.88 | 0.23 | 0.02 | 8.21 | 0.23 | 0.02 | 7.91 |
Yofm | g g−1 | 0.2795 | 0.20 | 0.01 | 4.74 | 0.20 | 0.02 | 7.53 | 0.22 | 0.01 | 5.46 | 0.21 | 0.02 | 9.87 | 0.23 | 0.01 | 5.75 | 0.21 | 0.02 | 10.21 | 0.22 | 0.01 | 5.20 | 0.22 | 0.01 | 5.08 |
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Hans, S.; Haby, B.; Krausch, N.; Barz, T.; Neubauer, P.; Cruz-Bournazou, M.N. Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations. Bioengineering 2020, 7, 145. https://doi.org/10.3390/bioengineering7040145
Hans S, Haby B, Krausch N, Barz T, Neubauer P, Cruz-Bournazou MN. Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations. Bioengineering. 2020; 7(4):145. https://doi.org/10.3390/bioengineering7040145
Chicago/Turabian StyleHans, Sebastian, Benjamin Haby, Niels Krausch, Tilman Barz, Peter Neubauer, and Mariano Nicolas Cruz-Bournazou. 2020. "Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations" Bioengineering 7, no. 4: 145. https://doi.org/10.3390/bioengineering7040145
APA StyleHans, S., Haby, B., Krausch, N., Barz, T., Neubauer, P., & Cruz-Bournazou, M. N. (2020). Automated Conditional Screening of Multiple Escherichia coli Strains in Parallel Adaptive Fed-Batch Cultivations. Bioengineering, 7(4), 145. https://doi.org/10.3390/bioengineering7040145