Comparison of light scattering signals vs BODIPY™ 493/503 staining in flow cytometry for the quantitative monitoring of P(3HB-co-3 HV) biosynthesis.

Abstract

Analysis of microbial cultures traditionally relies on average population characteristics, despite the inherent variability of individual cells. This variability affects process performance and makes bioprocesses scale-up challenging. Flow cytometry, a multi-parametric and near-line method provides information at cellular level. This study evaluated the potential of light scattering signals versus BODIPY™ 493/503 staining in flow cytometry for the quantitative monitoring of P(3HB-co-3 HV) biosynthesis by Cupriavidus necator. A dedicated bioreactor culture was performed to generate a dataset with samples presenting a wide range of P(3HB-co-3 HV) contents (6 to 70 wt%). The relationships were then validated using data from two independent cultures. For cells with levels ≥0.06 pg_PHA.cell^-1 (30 wt%) the FSC light scattering signal proved to be a simple fast and accurate method for monitoring phases of P(3HB-co-3 HV) accumulation without the need for staining. For early growth phases or low PHA contents (< 0.06 pg_PHA.cell^-1), BODIPY™ staining remains necessary to identify and quantify PHA-producing cells. Monitoring of sub-population dynamics during PHA production showed an increase in the diversity of cellular PHA content over time, with end-of-culture levels ranging from <0.01 pg_PHA.cell^-1 to over 0.86 pg_PHA.cell^-1, with damaged cells found mainly in subpopulations with low PHA content.