Adaptive laboratory evolution in a novel parallel shaken pH-auxostat

Area of Science:

• Biotechnology
• Microbial Engineering
• Synthetic Biology

Background:

• Adaptive laboratory evolution (ALE) is crucial for microbial strain development.
• Traditional ALE methods include batch or continuous cultivations, each with limitations.
• Combining high-throughput shaken cultures with continuous fermentation benefits ALE.

Purpose of the Study:

• To develop a novel Continuous parallel shaken pH-auxostat (CPA) system.
• To integrate advantages of parallel shaken cultures and continuous fermentation for ALE.
• To optimize ALE for rapid selection of improved microbial strains.

Main Methods:

• A CPA system with six autonomous shaken reactors and real-time pH control was developed.
• Noninvasive pH sensor spots and a programmable pump module controlled dilution rates.
• Two strains of *Ogataea polymorpha* were cultivated using chemostat and pH-auxostat methods.

Main Results:

• The CPA system enabled continuous cultivation with pH-controlled dilution rates near the washout point.
• Optimized PI controller parameters using the Chien-Hrones-Reswick (CHR) method.
• ALE in the CPA system selected for *O. polymorpha* strains with up to 4.8-fold increased maximum specific growth rates.

Conclusions:

• The CPA system effectively combines high throughput with continuous fermentation for ALE.
• This novel approach significantly accelerates the selection of faster-growing microbial strains.
• The developed method is valuable for microbial strain development and optimization.