Quantifying microbial robustness in dynamic environments using microfluidic single-cell cultivation.

BACKGROUND: Microorganisms must respond to changes in their environment. Analysing the robustness of functions (i.e. performance stability) to such dynamic perturbations is of great interest in both laboratory and industrial settings. Recently, a quantification method capable of assessing the robustness of various functions, such as specific growth rate or product yield, across different conditions, time frames, and populations has been developed for microorganisms grown in a 96-well plate. In micro-titer-plates, environmental change is slow and undefined. Dynamic microfluidic single-cell cultivation (dMSCC) enables the precise maintenance and manipulation of microenvironments, while tracking single cells over time using live-cell imaging. Here, we combined dMSCC and a robustness quantification method to a pipeline for assessing performance stability to changes occurring within seconds or minutes. RESULTS: Saccharomyces cerevisiae CEN.PK113-7D, harbouring a biosensor for intracellular ATP levels, was exposed to glucose feast-starvation cycles, with each condition lasting from 1.5 to 48 min over a 20 h period. A semi-automated image and data analysis pipeline was developed and applied to assess the performance and robustness of various functions at population, subpopulation, and single-cell resolution. We observed a decrease in specific growth rate but an increase in intracellular ATP levels with longer oscillation intervals. Cells subjected to 48 min oscillations exhibited the highest average ATP content, but the lowest stability over time and the highest heterogeneity within the population. CONCLUSION: The proposed pipeline enabled the investigation of function stability in dynamic environments, both over time and within populations. The strategy allows for parallelisation and automation, and is easily adaptable to new organisms, biosensors, cultivation conditions, and oscillation frequencies. Insights on the microbial response to changing environments will guide strain development and bioprocess optimisation.

Protocol to perform dynamic microfluidic single-cell cultivation of C. glutamicum.

Here, we present a protocol for the design, fabrication, and usage of a polydimethylsiloxane (PDMS)-based chip for dynamic microfluidic single-cell cultivation of Corynebacterium glutamicum. We describe steps for flow profile establishment and biological preparation. We then detail time-lapse imaging to observe reactions of C. glutamicum to repeated environmental changes in the range of seconds. This system can be adapted to other organisms with a cell wall and soluble non-gaseous environmental factors like nutrients. For complete details on the use and execution of this protocol, please refer to Täuber et al..1.

Microbial lifelines in bioprocesses: From concept to application.

Bioprocesses are scaled up for the production of large product quantities. With larger fermenter volumes, mixing becomes increasingly inefficient and environmental gradients get more prominent than in smaller scales. Environmental gradients have an impact on the microorganism's metabolism, which makes the prediction of large-scale performance difficult and can lead to scale-up failure. A promising approach for improved understanding and estimation of dynamics of microbial populations in large-scale bioprocesses is the analysis of microbial lifelines. The lifeline of a microbe in a bioprocess is the experience of environmental gradients from a cell's perspective, which can be described as a time series of position, environment and intracellular condition. Currently, lifelines are predominantly determined using models with computational fluid dynamics, but new technical developments in flow-following sensor particles and microfluidic single-cell cultivation open the door to a more interdisciplinary concept. We critically review the current concepts and challenges in lifeline determination and application of lifeline analysis, as well as strategies for the integration of these techniques into bioprocess development. Lifelines can contribute to a successful scale-up by guiding scale-down experiments and identifying strain engineering targets or bioreactor optimisations.

Coupling Cell Size Regulation and Proliferation Dynamics of C. glutamicum Reveals Cell Division Based on Surface Area.

Single cells actively coordinate growth and division to regulate their size, yet how this size homeostasis at the single-cell level propagates over multiple generations to impact clonal expansion remains fundamentally unexplored. Classical timer models for cell proliferation (where the duration of the cell cycle is an independent variable) predict that the stochastic variation in colony size will increase monotonically over time. In stark contrast, implementing size control according to adder strategy (where on average a fixed size added from cell birth to division) leads to colony size variations that eventually decay to zero. While these results assume a fixed size of the colony-initiating progenitor cell, further analysis reveals that the magnitude of the intercolony variation in population number is sensitive to heterogeneity in the initial cell size. We validate these predictions by tracking the growth of isogenic microcolonies of Corynebacterium glutamicum in microfluidic chambers. Approximating their cell shape to a capsule, we observe that the degree of random variability in cell size is different depending on whether the cell size is quantified as per length, surface area, or volume, but size control remains an adder regardless of these size metrics. A comparison of the observed variability in the colony population with the predictions suggests that proliferation matches better with a cell division based on the cell surface. In summary, our integrated mathematical-experimental approach bridges the paradigms of single-cell size regulation and clonal expansion at the population levels. This innovative approach provides elucidation of the mechanisms of size homeostasis from the stochastic dynamics of colony size for rod-shaped microbes.

Microfluidic single-cell scale-down bioreactors: A proof-of-concept for the growth of Corynebacterium glutamicum at oscillating pH values.

In large-scale bioreactors, gradients in cultivation parameters such as oxygen, substrate, and pH result in fluctuating cell environments. pH fluctuations were identified as a critical parameter for bioprocess performance. Traditionally, scale-down systems at the laboratory scale are used to analyze the effects of fluctuating pH values on strains and thus process performance. Here, we demonstrate the application of dynamic microfluidic single-cell cultivation (dMSCC) as a novel scale-down system for the characterization of Corynebacterium glutamicum growth using oscillating pH conditions as a model stress factor. A detailed comparison between two-compartment reactor (two-CR) scale-down experiments and dMSCC was performed for one specific pH oscillation between reference pH 7 (~8 min) and disturbed pH 6 (~2 min). Similar reductions in growth rates were observed in both systems (dMSCC 21% and two-CR 27%) compared to undisturbed cultivation at pH 7. Afterward, systematic experiments at symmetric and asymmetric pH oscillations, between pH ranges of 4-6 and 8-11 and different intervals from 1 to 20 min, were performed to demonstrate the unique application range and throughput of the dMSCC system. Finally, the strength of the dMSCC application was demonstrated by mimicking fluctuating environmental conditions of a putative large-scale bioprocess, which is difficult to conduct using two-CRs.

How to Perform a Microfluidic Cultivation Experiment-A Guideline to Success.

As a result of the steadily ongoing development of microfluidic cultivation (MC) devices, a plethora of setups is used in biological laboratories for the cultivation and analysis of different organisms. Because of their biocompatibility and ease of fabrication, polydimethylsiloxane (PDMS)-glass-based devices are most prominent. Especially the successful and reproducible cultivation of cells in microfluidic systems, ranging from bacteria over algae and fungi to mammalians, is a fundamental step for further quantitative biological analysis. In combination with live-cell imaging, MC devices allow the cultivation of small cell clusters (or even single cells) under defined environmental conditions and with high spatio-temporal resolution. Yet, most setups in use are custom made and only few standardised setups are available, making trouble-free application and inter-laboratory transfer tricky. Therefore, we provide a guideline to overcome the most frequently occurring challenges during a MC experiment to allow untrained users to learn the application of continuous-flow-based MC devices. By giving a concise overview of the respective workflow, we give the reader a general understanding of the whole procedure and its most common pitfalls. Additionally, we complement the listing of challenges with solutions to overcome these hurdles. On selected case studies, covering successful and reproducible growth of cells in MC devices, we demonstrate detailed solutions to solve occurring challenges as a blueprint for further troubleshooting. Since developer and end-user of MC devices are often different persons, we believe that our guideline will help to enhance a broader applicability of MC in the field of life science and eventually promote the ongoing advancement of MC.

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

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.

Production of Food and Feed Additives From Non-food-competing Feedstocks: Valorizing N-acetylmuramic Acid for Amino Acid and Carotenoid Fermentation With Corynebacterium glutamicum.

Corynebacterium glutamicum is used for the million-ton-scale production of food and feed amino acids such as L-glutamate and L-lysine and has been engineered for production of carotenoids such as lycopene. These fermentation processes are based on sugars present in molasses and starch hydrolysates. Due to competing uses of starch and sugars in human nutrition, this bacterium has been engineered for utilization of alternative feedstocks, for example, pentose sugars present in lignocellulosic and hexosamines such as glucosamine (GlcN) and N-acetyl-D-glucosamine (GlcNAc). This study describes strain engineering and fermentation using N-acetyl-D-muramic acid (MurNAc) as non-food-competing feedstock. To this end, the genes encoding the MurNAc-specific PTS subunits MurP and Crr and the etherase MurQ from Escherichia coli K-12 were expressed in C. glutamicumΔnanR. While MurP and MurQ were required to allow growth of C. glutamicumΔnanR with MurNAc, heterologous Crr was not, but it increased the growth rate in MurNAc minimal medium from 0.15 h-1 to 0.20 h-1. When in addition to murP-murQ-crr the GlcNAc-specific PTS gene nagE from C. glycinophilum was expressed in C. glutamicumΔnanR, the resulting strain could utilize blends of GlcNAc and MurNAc. Fermentative production of the amino acids L-glutamate and L-lysine, the carotenoid lycopene, and the L-lysine derived chemicals 1,5-diaminopentane and L-pipecolic acid either from MurNAc alone or from MurNAc-GlcNAc blends was shown. MurNAc and GlcNAc are the major components of the bacterial cell wall and bacterial biomass is an underutilized side product of large-scale bacterial production of organic acids, amino acids or enzymes. The proof-of-concept for valorization of MurNAc reached here has potential for biorefinery applications to convert non-food-competing feedstocks or side-streams to valuable products such as food and feed additives.