4 SPECIES OF BACTERIA DETERMINISTICALLY FORM A STABLE BIOFILM IN A MILLIFLUIDIC CHANNEL: ASSEMBLY PRINCIPLES

A bstract Multispecies microbial adherent communities are widespread in nature and organisms but the principles of their assembly and development remain unclear. Yet, the demand to understand and predict the responses of such living communities to environmental changes is increasing, calling for new approaches. Here, we test the possibility to establish a simplified but relevant model of multispecies biofilm in a laboratory setup enabling in situ real-time monitoring of the community development and control of the environmental parameters in order to decipher the mechanisms underlying the formation of the community. Using video-microscopy and species combinatorial approach, we assess the global and individual species spatiotemporal development in millifluidic channels under constant flow of nutrients. Based on quantitative measurements of expansion kinetics, local dynamics and spatial distribution, we demonstrate that the four chosen species ( Bacillus thuringiensis, Pseudomonas fluorescens, Kocuria varians and Rhodocyclus sp.) form a dynamical community that deterministically reaches its equilibrium after about 30 hours of growth. We evidence the emergence of complexity in this simplified community as reported by spatial heterogeneity rise and non-monotonic developmental kinetics. We find interspecies interactions consisting in competition for resources — in particular oxygen — and both direct and indirect physical interactions but no positive feedback. Thereby, we introduce a model of multispecies adherent community where effective couplings result from individual species quest for fitness optimization in a moving and heterogenous environment. This control and the understanding of this simplified experimental model shall open new avenues to apprehend adherent bacterial communities behavior in a context of rapid global change.