Unveiling microbial single-cell growth dynamics under rapid periodic oxygen oscillations

Microbial metabolism and growth are tightly linked to oxygen (O2). Microbes experience fluctuating O2 levels in natural environments; however, our understanding of how cells respond to fluctuating O2 over various time scales remains limited, due to challenges in observing microbial growth at single-cell resolution under controlled O2 conditions and in linking individual cell growth with the specific O2 microenvironment. We performed time-resolved microbial growth analyses at single-cell resolution under a temporally controlled O2 supply. A multilayer microfluidic device was developed, featuring gas supply above a cultivation layer, separated by a thin membrane enabling efficient gas transfer. This platform enables microbial cultivation under constant, dynamic, and oscillating O2 conditions. Automated time-lapse microscopy and deep-learning-based image analysis provide access to spatiotemporally resolved growth data at the single-cell level. O2 switching within tens of seconds, coupled with precise monitoring of the microenvironment, allows us to accurately correlate cellular growth with local O2 concentrations. Growing Escherichia coli microcolonies subjected to varying O2 oscillation periods show distinct growth dynamics, characterized by response and recovery phases. The comprehensive growth data and insights gained from our unique platform are a crucial step forward to systematically study cell response and adaptation to fluctuating O2 environments at single-cell resolution.