Flow cytometry-based viability staining: an at-line tool for bioprocess monitoring of Sulfolobus acidocaldarius

Abstract

Determination of the viability, ratio of dead and live cell populations, of Sulfolobus acidocaldarius is still being done by tedious and material-intensive plating assays that can only provide time-lagged results. Although S. acidocaldarius, an extremophilic Archaeon thriving at 75 °C and pH 3.0, and related species harbor great potential for the exploitation as production hosts and biocatalysts in biotechnological applications, no industrial processes have been established yet. One hindrance is that during development and scaling of industrial bioprocesses timely monitoring of the impact of process parameters on the cultivated organism is crucial-a task that cannot be fulfilled by traditional plating assays. As alternative, flow cytometry (FCM) promises a fast and reliable method for viability assessment via the use of fluorescent dyes. In this study, commercially available fluorescent dyes applicable in S. acidocaldarius were identified. The dyes, fluorescein diacetate and concanavalin A conjugated with rhodamine, were discovered to be suitable for viability determination via FCM. For showing the applicability of the developed at-line tool for bioprocess monitoring, a chemostat cultivation on a defined growth medium at 75 °C, pH 3.0 was conducted. Over the timeframe of 800 h, this developed FCM method was compared to the plating assay by monitoring the change in viability upon controlled pH shifts. Both methods detected an impact on the viability at pH values of 2.0 and 1.5 when compared to pH 3.0. A logarithmic relationship between the viability observed via plating assay and via FCM was observed.

Keywords: Flow cytometry; Fluorescent dyes; Live/dead staining; Sulfolobus acidocaldarius; Viability.

Fig. 1

Gate definition for viability evaluation of Sulfolobus acidocaldarius. A density plot of side scatter versus forward scatter for ConA-rhodamine in PBS buffer, showing the background, color code: red-high to purple-low; B density plot of side scatter versus forward scatter for cells stained with FDA and ConA-rhodamine, showing the cell gate; C density plot of FL1 (536/40 nm bandpass) versus FL4 (610/30 nm bandpass) of cells gated in B; D statistics of FL1 vs. FL4 shown in C

Fig. 2

A Sensitivity analysis of viability according to mixed ratios [%] vs. V_FCM [%]. Viability according to mixed ratios [%] were obtained by mixing different ratios of non-viable and viable cell populations. V_FCM cells [%] were measured by the flow cytometer and evaluated according to Fig. 1. B Comparison of state-of-the-art method log[V_plating (CFU/mL/OD₆₀₀)] versus V_FCM [%]

Fig. 3

Comparison of viability measurements of Sulfolobus acidocaldarius in response to the shift in pH value, observed over time [h]. The viability was determined by flow cytometry (V_FCM [%]) and by plating assay (V_plating [%]). Both pH shifts from 3.0 to 2.0 and to 1.5, respectively caused a drop in both viability-determining methods as well as in OD₆₀₀. In this case, V_plating measured at the beginning of the experiment was set to 100%, and for determining V_plating [%], the CFUs of each sample were then divided by this initial value

Fig. 4

Logarithmic trend of V_FCM [%] versus log (V_plating) with a correlation factor of R² = 0.90