Real-time spatial O2-sensing in 2D porous media to investigate conditions for anoxic micro-niches onset and microbial facultative metabolism trigger.

The heterogeneous spatial organization and composition of bacterial communities in soils are expected to deeply impact geochemical quantities at the pore-scale, shaping steep solutes gradients in spite of mixing processes. Oxygen distribution is of major interest since localized anoxic niches might host facultatively anaerobic bacteria. This means that metabolisms alternative to aerobic respiration might be triggered even in well-oxygenated systems, thus largely impacting soil ecological functions.

Our ability to non-invasively monitor O2 time-space distributions at the scale of interest for bacterial clusters is still limited. Moreover, the identification of the critical O2 level at which facultative bacteria switch their metabolism is little explored and largely debated. 

This work presents an innovative experimental setup allowing to simultaneously quantify oxygen concentrations and biomass arrangement as a function of time and space in microfluidic devices, which mimic natural porous media. Our methodology makes use of i) a newly developed and customized planar transparent fluorescent sensor whose fluorescence intensity is sensitive to oxygen concentration, and ii) a fully automated microscope to collect high-resolution large images.

Our results reveal that microbial aggregates and oxygen distribution are closely correlated both spatially and temporally demonstrating that microbial activity can generate and sustain anoxic micro-niches. This anoxic space occupies up to 2-3 % of the porous volume and is controlled by the competition between advective/diffusive processes (supplying oxygen) and microbial O2 consumption. Interestingly, bacterial cluster shapes and their spatial organization are key elements determining the development of anoxic micro-niches and their impact on macroscale processes. Based on the new insights provided by our experiment, we develop an original definition of Damköhler number to describe the conditions for anoxic micro-niches onset under the laminar diffusion-dominated flow that typically characterized groundwater systems.

This novel methodology combined with opportunely tagged laboratory strains opens new frontiers to investigate the O2 critical concentration associated with facultative metabolism trigger for bacteria with interesting function in soil ecology and wastewater water and drinking water remediation engineering.