Development of two devices for high-throughput screening of ethanol-producing microorganisms by real-time CO2 production monitoring

Abstract

Bioethanol's importance as a renewable energy carrier led to the development of new devices for the high-throughput screening (HTS) of ethanol-producing microorganisms, monitoring ethanol production, and process optimization. This study developed two devices based on measuring CO₂ evolution (an equimolar byproduct of microbial ethanol fermentation) to allow for a fast and robust HTS of ethanol-producing microorganisms for industrial purposes. First, a pH-based system for identifying ethanol producers (Ethanol-HTS) was established in a 96-well plate format where CO₂ emission is captured by a 3D-printed silicone lid and transferred from the fermentation well to a reagent containing bromothymol blue as a pH indicator. Second, a self-made CO₂ flow meter (CFM) was developed as a lab-scale tool for real-time quantification of ethanol production. This CFM contains four chambers to simultaneously apply different fermentation treatments while LCD and serial ports allow fast and easy data transfer. Applying ethanol-HTS with various yeast concentrations and yeast strains displayed different colors, from dark blue to dark and light green, based on the amount of carbonic acid formed. The results of the CFM device revealed a fermentation profile. The curve of CO₂ production flow among six replications showed the same pattern in all batches. The comparison of final ethanol concentrations calculated based on CO₂ flow by the CFM device with the GC analysis showed 3% difference which is not significant. Data validation of both devices demonstrated their applicability for screening novel bioethanol-producer strains, determining carbohydrate fermentation profiles, and monitoring ethanol production in real time.

Keywords: Bioethanol; CO2 measurement; High-throughput screening; Real-time analysis; pH indicators.