Developing an Automated System for Yeast Culture Cultivation and Control using Flow Cytometry

Sammanfattning: Flow cytometry is a versatile tool for monitoring a microbial population at a single-cell level. Multiple parameters can be monitored based on the users’ requirements through the utilization of different dyes, biosensors etc., and the use of FCM for on-line measurements during fed-batch cultivations has the potential to be used for precise process control. This project consisted of the development and testing of a Python program for on-line bioprocess regulation based on flow cytometry data. Specifically, the program was built to process FCS files resulting from at-line measurement of pentose-fermenting S. cerevisiae using the OnCyt autosampler connected to a BD Accuri C6+ flow cytometer. The program was designed to automatically start and stop a peristaltic pump through serial communication, by issuing commands based on changes within the microbial population. The yeast strain used was the TMBRP011 strain, which was previously engineered for bioethanol production from pentose sugars and carries a previously developed redox biosensor. The biosensor reacts to cellular redox imbalance caused by inhibitory substances released during lignocellulose pretreatments through an increase in fluorescence. The program was designed to achieve higher volumes of inhibitory substances as the cells acclimatized to a set rate, indicated with a decrease in fluorescence. However, since these inhibitory substances are toxic in nature, the pump should stop pumping if the percentage of PI-stained cells within a sample increased, indicating cell membrane damage. The program was successful in regulating a fed-batch process based on the prementioned parameters, and it managed to reinduce the cells by injecting higher volumes of inhibitory compounds per hour. The program was developed in a way that fulfilled the initial scope in terms of functionality and provides a roadmap for implementing on-line FCM as a basis for process control.