Microbial single-cell growth response at defined carbon limiting conditions

Lindemann, Dorina; Westerwalbesloh, Christoph; Kohlheyer, Dietrich; Grünberger, Alexander; Lieres, Eric von

doi:10.1039/c9ra02454a

Cited by 17 publications

(19 citation statements)

References 54 publications

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“…In summary, the reduction of PCA concentration mainly affected growth rate and batch time, but as a general principle, lowering the concentration below critical thresholds typically resulted in significantly reduced reproducibility between biological replicates. This observation is supported by current reports from Lindemann et al whose microfluidic cultivation experiments with C. glutamicum growing on PCA as sole carbon source revealed, besides other effects, significant influence of PCA concentration on doubling time and cell-to-cell variability [25]. Anyway, for the given process, batch time proved to be the most sensitive parameter with respect to PCA variation.…”

Section: Protocatechuic Acid Interferes With Ph Measurementsupporting

confidence: 78%

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

Morschett

Jansen

Neuendorf

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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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.

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Section: Protocatechuic Acid Interferes With Ph Measurementsupporting

confidence: 78%

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

Morschett

Jansen

Neuendorf

et al. 2020

Journal of Industrial Microbiology and Biotechnology

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“…The current state of knowledge related to the C. glutamicum response to pH stress is based on bulk population studies with microbial cells cultivated in small bioreactors or shaking flasks (Jakob et al, 2007;Follmann et al, 2009b). Using these methods, representative information cannot be gathered for individual cells (Lindström and Andersson-Svahn, 2010), and cell-tocell heterogeneity remains unclear (Lindemann et al, 2019). Furthermore, traditional cultivation lacks temporal precision and spatial resolution, e.g., due to slow mixing in large volumes to perform stress response experiments, such as those with defined stress pulses, or even the investigation of oscillating stress conditions (Lara et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Growth Response and Recovery of Corynebacterium glutamicum Colonies on Single-Cell Level Upon Defined pH Stress Pulses

et al. 2021

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Bacteria respond to pH changes in their environment and use pH homeostasis to keep the intracellular pH as constant as possible and within a small range. A change in intracellular pH influences enzyme activity, protein stability, trace element solubilities and proton motive force. Here, the species Corynebacterium glutamicum was chosen as a neutralophilic and moderately alkali-tolerant bacterium capable of maintaining an internal pH of 7.5 ± 0.5 in environments with external pH values ranging between 5.5 and 9. In recent years, the phenotypic response of C. glutamicum to pH changes has been systematically investigated at the bulk population level. A detailed understanding of the C. glutamicum cell response to defined short-term pH perturbations/pulses is missing. In this study, dynamic microfluidic single-cell cultivation (dMSCC) was applied to analyze the physiological growth response of C. glutamicum to precise pH stress pulses at the single-cell level. Analysis by dMSCC of the growth behavior of colonies exposed to single pH stress pulses (pH = 4, 5, 10, 11) revealed a decrease in viability with increasing stress duration w. Colony regrowth was possible for all tested pH values after increasing lag phases for which stress durations w were increased from 5 min to 9 h. Furthermore, single-cell analyses revealed heterogeneous regrowth of cells after pH stress, which can be categorized into three physiological states. Cells in the first physiological state continued to grow without interruption after pH stress pulse. Cells in the second physiological state rested for several hours after pH stress pulse before they started to grow again after this lag phase, and cells in the third physiological state did not divide after the pH stress pulse. This study provides the first insights into single-cell responses to acidic and alkaline pH stress by C. glutamicum.

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“…Contrary to studies in bioreactors, it is possible to follow phenotypic development and regulation of isolated cells with spatiotemporal resolution and to distinguish contributions from intrinsic stochastic processes and environmental factors (Weibel et al, 2007;Dusny and Schmid, 2015). On this basis, the systems can reveal fundamental insight into cellular regulation strategies to nutrient-limited conditions (Lindemann et al, 2019). Several microfluidic cultivation concepts exist where cells are trapped by different physical principles.…”

Section: Discussion Of Single-cell Technologies For the Study Of Adapmentioning

confidence: 99%

Single-Cell Technologies to Understand the Mechanisms of Cellular Adaptation in Chemostats

Wright

Rønnest

Sonnenschein

2020

Front. Bioeng. Biotechnol.

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There is a growing interest in continuous manufacturing within the bioprocessing community. In this context, the chemostat process is an important unit operation. The current application of chemostat processes in industry is limited although many high yielding processes are reported in literature. In order to reach the full potential of the chemostat in continuous manufacture, the output should be constant. However, adaptation is often observed resulting in changed productivities over time. The observed adaptation can be coupled to the selective pressure of the nutrient-limited environment in the chemostat. We argue that population heterogeneity should be taken into account when studying adaptation in the chemostat. We propose to investigate adaptation at the single-cell level and discuss the potential of different single-cell technologies, which could be used to increase the understanding of the phenomena. Currently, none of the discussed single-cell technologies fulfill all our criteria but in combination they may reveal important information, which can be used to understand and potentially control the adaptation.

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Microbial single-cell growth response at defined carbon limiting conditions

Abstract: Using microfluidic single-cell cultivation technologies and modelling we examined how single-cell growth at defined carbon conditions, ranging from strongly limiting conditions to a carbon surplus, influenced cell-to-cell variability.

Cited by 17 publications

References 54 publications

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

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

Growth Response and Recovery of Corynebacterium glutamicum Colonies on Single-Cell Level Upon Defined pH Stress Pulses

Single-Cell Technologies to Understand the Mechanisms of Cellular Adaptation in Chemostats

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