Estimation of cell viability with high-resolution growth curves

Abstract

Fuel grade bioethanol is produced through distillation of a liquid product which comes from fermentation of the sugars constituting biomass (Petrou, 2009). However, fermentation of lignocellulosic materials presents some challenges, one of them being the toxicity of the substrate for the fermenting organism. Toxic effects of lignocellulosic raw material inhibitors and their effect on the metabolism and viability of the fermenting organism is a major research subject. The work presented in this report aimed at developing a superior method for the determination of the fraction of viable cells in a yeast population. The method is based on growth curve determinations performed with the Bioscreen CMBR instrument. This equipment is a tool for microbiology optimized for growth analyzes (Bioscreen, 2011). Experiments carried out with Bioscreen allowed developing a method to perform optimal growth curves in this equipment after some experimental parameters were optimized. The best ones are continuous shaking with high intensity and initial optical density = 0.10. The temperature and the volume were set to 30 ° and 145 µl, respectively (Murakami, 2009). In order to estimate the number of viable cells, several growth curves with different percentages of viable cells were performed in Bioscreen. Doing the comparison between two growth curves with different percentages of viable cells, it is possible to observe a "delay time" to reach a target OD. Based on this delay, it is possible to estimate the number of viable cells presented in the studied population. Analyzing the data from the experiments with mixed populations with different percentage of viable cells it was possible to develop a method to estimate the number of viable cells in a sample. However, it was possible to successfully apply this method only for populations with percentage of viable cells between 70 and 100%. Suggestions on future perspectives and strategies to possibly improve the method are presented.