Physico-chemical regulation of bacterial growth success under 3D confinement

How environmental factors influence bacterial growth remains an extensively pursued, yet perennially challenging question. Most natural bacterial habitats are complex 3D spaces, featuring disordered spatial architectures, heterogeneous nutrient availability, and broad temperature ranges. However, state-of-the-art knowledge on bacterial growth almost entirely derives from conventional experimental platforms - liquid culture and 2D flat plates - which offer limited capacity to recapitulate the diverse physico-chemical aspects intrinsic to physiological niches. Consequently, we do not fully understand how manifold environmental variables act in concert to influence growth dynamics. Here, we report the first-such interrogation of bacterial growth success under 3D confinement across combinatorial physico-chemical regimes. We accomplish this by engineering mechanically tunable viscoelastic 3D porous media, multiplexed with three distinct nutrient compositions and five different growth temperatures. Across this parameter space, we discover regimes of either confinement-driven or temperature-driven growth success, as well as nutrient composition-dependent modulation of these effects. Our work identifies a tripartite regulatory framework that captures the inter-linked effects of three generic environmental variables - mechanical constraints, nutrient availability, and temperature - on growth under 3D confinement, supported by direct experimental evidence. Together, our findings establish how environmental factors influence growth across physico-chemically complex 3D milieus.