Asymmetric division of Hansenula polymorpha reflected by a drop of light scatter intensity measured in batch microtiter plate cultivations at phosphate limitation

Abstract:
Hansenula polymorpha RB11 pC10-FMD (PFMD
GFP) (FMD promoter gfp gene) was simultaneously cultivated in the Respiration Activity Monitoring System (RAMOS) and in the microtiter plate cultivation system ‘‘BioLector’’ under phosphate limitation. The light scatter signal of the BioLector, for the determination of the biomass concentration in the wells, shows a significant decrease with the onset of the phosphate limitation until a stationary level is reached. At lower initial phosphate concentration this effect is more pronounced and longer time is required until the stationary level of the scattered light is achieved. The oxygen transfer rate signal of the RAMOS and the light scatter signal of the BioLector correlate with respect to the points of time where the maxima and the stationary levels of the courses are reached. In order to understand the effect causing this light scatter behavior, the forward and side scatter properties were investigated off line by flow cytometry. The decay in the light scatter of the BioLector seems to correlate with the formation of two subpopulations of different scatter intensities detected by a flow cytometer. With ongoing cultivation the fraction of cells possessing higher light scattering properties decreases until only a population of lower light scattering properties exists. The rate of transition of the yeast from one subpopulation to the other appears to be correlated with the rate of decrease in the BioLector light scatter signal. The formation of the subpopulations may be caused by an increased asymmetry in the cell cycle due to phosphate limitation.

Keywords:
respiration activity monitoring system, bio- lector, Hansenula polymorpha, flow cytometry, phosphate limitation, process development

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