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Parallelized microscale fed-batch cultivation in online-monitored microtiter plates: implications of media composition and feed strategies for process design and performance.

ABSTRACT: Limited throughput represents a substantial drawback during bioprocess development. In recent years, several commercial microbioreactor systems have emerged featuring parallelized experimentation with optical monitoring. However, many devices remain limited to batch mode and do not represent the fed-batch strategy typically applied on an industrial scale. A workflow for 32-fold parallelized microscale cultivation of protein secreting Corynebacterium glutamicum in microtiter plates incorporating online monitoring, pH control and feeding was developed and validated. Critical interference of the essential media component protocatechuic acid with pH measurement was revealed, but was effectively resolved by 80% concentration reduction without affecting biological performance. Microfluidic pH control and feeding (pulsed, constant and exponential) were successfully implemented: Whereas pH control improved performance only slightly, feeding revealed a much higher optimization potential. Exponential feeding with µ = 0.1 h-1 resulted in the highest product titers. In contrast, other performance indicators such as biomass-specific or volumetric productivity resulted in different optimal feeding regimes.

SUBMITTER: Morschett H

PROVIDER: S-EPMC6971147 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Parallelized microscale fed-batch cultivation in online-monitored microtiter plates: implications of media composition and feed strategies for process design and performance.

Morschett Holger H Jansen Roman R Neuendorf Christian C Moch Matthias M Wiechert Wolfgang W Oldiges Marco M

Journal of industrial microbiology & biotechnology 20191031 1

Limited throughput represents a substantial drawback during bioprocess development. In recent years, several commercial microbioreactor systems have emerged featuring parallelized experimentation with optical monitoring. However, many devices remain limited to batch mode and do not represent the fed-batch strategy typically applied on an industrial scale. A workflow for 32-fold parallelized microscale cultivation of protein secreting Corynebacterium glutamicum in microtiter plates incorporating ...[more]

PMID: 31673873

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Project description:BackgroundSubstrate-limited fed-batch conditions have the favorable effect of preventing overflow metabolism, catabolite repression, oxygen limitation or inhibition caused by elevated substrate or osmotic concentrations. Due to these favorable effects, fed-batch mode is predominantly used in industrial production processes. In contrast, screening processes are usually performed in microtiter plates operated in batch mode. This leads to a different physiological state of the production organism in early screening and can misguide the selection of potential production strains. To close the gap between screening and production conditions, new techniques to enable fed-batch mode in microtiter plates have been described. One of these systems is the ready-to-use and disposable polymer-based controlled-release fed-batch microtiter plate (fed-batch MTP). In this work, the fed-batch MTP was applied to establish a glucose-limited fed-batch screening procedure for industrially relevant protease producing Bacillus licheniformis strains.ResultsTo achieve equal initial growth conditions for different clones with the fed-batch MTP, a two-step batch preculture procedure was developed. Based on this preculture procedure, the standard deviation of the protease activity of glucose-limited fed-batch main culture cultivations in the fed-batch MTP was ± 10%. The determination of the number of replicates revealed that a minimum of 6 parallel cultivations were necessary to identify clones with a statistically significant increased or decreased protease activity. The developed glucose-limited fed-batch screening procedure was applied to 13 industrially-relevant clones from two B. licheniformis strain lineages. It was found that 12 out of 13 clones (92%) were classified similarly as in a lab-scale fed-batch fermenter process operated under glucose-limited conditions. When the microtiter plate screening process was performed in batch mode, only 5 out of 13 clones (38%) were classified similarly as in the lab-scale fed-batch fermenter process.ConclusionThe glucose-limited fed-batch screening process outperformed the usual batch screening process in terms of the predictability of the clone performance under glucose-limited fed-batch fermenter conditions. These results highlight that the implementation of glucose-limited fed-batch conditions already in microtiter plate scale is crucial to increase the precision of identifying improved protease producing B. licheniformis strains. Hence, the fed-batch MTP represents an efficient high-throughput screening tool that aims at closing the gap between screening and production conditions.

Project description:BackgroundSmall-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved.ResultsCustomization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24-0.90 mg/h per well.ConclusionsThe optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates.

Project description:BackgroundFed-batch conditions are advantageous for industrial cultivations as they avoid unfavorable phenomena appearing in batch cultivations. Those are for example the formation of overflow metabolites, catabolite repression, oxygen limitation or inhibition due to elevated osmotic concentrations. For both, the early bioprocess development and the optimization of existing bioprocesses, small-scale reaction vessels are applied to ensure high throughput, low costs and prompt results. However, most conventional small-scale procedures work in batch operation mode, which stands in contrast to fed-batch conditions in large-scale bioprocesses. Extensive expenditure for installations and operation accompany almost all cultivation systems in the market allowing fed-batch conditions in small-scale. An alternative, more cost efficient enzymatic glucose release system is strongly influenced by environmental conditions. To overcome these issues, this study investigates a polymer-based fed-batch system for controlled substrate release in microtiter plates.ResultsImmobilizing a solid silicone matrix with embedded glucose crystals at the bottom of each well of a microtiter plate is a suitable technique for implementing fed-batch conditions in microtiter plates. The results showed that the glucose release rate depends on the osmotic concentration, the pH and the temperature of the medium. Moreover, the applied nitrogen source proved to influence the glucose release rate. A new developed mathematical tool predicts the glucose release for various media conditions. The two model organisms E. coli and H. polymorpha were cultivated in the fed-batch microtiter plate to investigate the general applicability for microbial systems. Online monitoring of the oxygen transfer rate and offline analysis of substrate, product, biomass and pH confirmed that fed-batch conditions are comparable to large-scale cultivations. Furthermore, due to fed-batch conditions in microtiter plates, product formation could be enhanced by the factor 245 compared to batch cultivations.ConclusionsThe polymer-based fed-batch microtiter plate represents a sophisticated and cost efficient system to mimic typical industrial fed-batch conditions in small-scale. Thus, a more reliable strain screening and early process development can be performed. A systematical scale-down with low expenditure of work, time and money is possible.

Dataset Information

Parallelized microscale fed-batch cultivation in online-monitored microtiter plates: implications of media composition and feed strategies for process design and performance.

Publications

Parallelized microscale fed-batch cultivation in online-monitored microtiter plates: implications of media composition and feed strategies for process design and performance.

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