Single cell analysis applied to antibody fragment production with Bacillus megaterium: development of advanced physiology and bioprocess state estimation tools

Abstract

BackgroundSingle cell analysis for bioprocess monitoring is an important tool to gain deeper insights into particular cell behavior and population dynamics of production processes and can be very useful for discrimination of the real bottleneck between product biosynthesis and secretion, respectively.ResultsHere different dyes for viability estimation considering membrane potential (DiOC2(3), DiBAC4(3), DiOC6(3)) and cell integrity (DiBAC4(3)/PI, Syto9/PI) were successfully evaluated for Bacillus megaterium cell characterization. It was possible to establish an appropriate assay to measure the production intensities of single cells revealing certain product secretion dynamics. Methods were tested regarding their sensitivity by evaluating fluorescence surface density and fluorescent specific concentration in relation to the electronic cell volume. The assays established were applied at different stages of a bioprocess where the antibody fragment D1.3 scFv production and secretion by B. megaterium was studied.ConclusionsIt was possible to distinguish between live, metabolic active, depolarized, dormant, and dead cells and to discriminate between high and low productive cells. The methods were shown to be suitable tools for process monitoring at single cell level allowing a better process understanding, increasing robustness and forming a firm basis for physiology-based analysis and optimization with the general application for bioprocess development.

Microbial heterogeneity affects bioprocess robustness: Dynamic single‐cell analysis contributes to understanding of microbial populations

“…In this case, the protein of interest can be tagged by immunofluorescence staining directly at the cell surface and easily detected by FC. David et al [67] used FC for analyzing cell viability and secretory capacity for the production of antibody fragment by Bacillus megaterium. Protein secretion was followed by staining the products attached to the cells when exported by membrane-coupled secretory (Sec) pathway.…”

A flow cytometric method for viability assessment of Staphylococcus aureus and Burkholderia cepacia in mixed culture

“…This may be caused by PI entry mediated by cell wall perforation encountered during cell division and cell wall synthesis (15,47), especially for exponentially growing cells. Concomitantly, the frequency of damaged S. aureus cells increased.…”

“…Concomitantly, the frequency of damaged S. aureus cells increased. This may be caused by PI entry mediated by cell wall perforation encountered during cell division and cell wall synthesis (15,47), especially for exponentially growing cells. Accordingly, PI uptake in highly reproductive bacteria during exponential growth was reported in several studies (47,48).…”

“…This may be caused by PI entry mediated by cell wall perforation encountered during cell division and cell wall synthesis (15,47), especially for exponentially growing cells. Accordingly, PI uptake in highly reproductive bacteria during exponential growth was reported in several studies (47,48). Furthermore, some of the events here classified as damaged might be associated with aggregation of viable and dead S. aureus cells, since it had previously been shown that Staphylococci clump during growth (49).…”

Monitoring of population dynamics of Corynebacterium glutamicum by multiparameter flow cytometry

“…Previous studies revealed that even simple parameters as the cell size, granularity or DNA content give already valuable insights into the metabolic status and the fitness of the whole population (Erlebach et al ., ; Hewitt and Nebe‐von‐Caron, ; Müller, ). Membrane potential (MP) and integrity are further parameters, which can be analysed by the use of specific fluorescent dyes and do provide important information regarding the cellular stress response and viability parameters, which were shown in several studies to significantly correlate with productivity and economic efficiency of the bioprocess (Hewitt et al ., ; David et al ., ). When equipped with a cell sorting capability for separation of single bacterial cells, FC analyses can be complemented with further downstream investigations, such as microscopy, cultivation in liquid or on agar plates, or even Omics ‐based technologies (Bernas et al ., ; Wiacek et al ., ; Jehmlich et al ., ).…”