Stochastic Assembly of Bacteria in Microwell Arrays Reveals the Importance of Confinement in Community Development

Abstract

The structure and function of microbial communities is deeply influenced by the physical and chemical architecture of the local microenvironment and the abundance of its community members. The complexity of this natural parameter space has made characterization of the key drivers of community development difficult. In order to facilitate these characterizations, we have developed a microwell platform designed to screen microbial growth and interactions across a wide variety of physical and initial conditions. Assembly of microbial communities into microwells was achieved using a novel biofabrication method that exploits well feature sizes for control of innoculum levels. Wells with incrementally smaller size features created populations with increasingly larger variations in inoculum levels. This allowed for reproducible growth measurement in large (20 μm diameter) wells, and screening for favorable growth conditions in small (5, 10 μm diameter) wells. We demonstrate the utility of this approach for screening and discovery using 5 μm wells to assemble P. aeruginosa colonies across a broad distribution of innoculum levels, and identify those conditions that promote the highest probability of survivial and growth under spatial confinement. Multi-member community assembly was also characterized to demonstrate the broad potential of this platform for studying the role of member abundance on microbial competition, mutualism and community succession.

Development of transparent microwell arrays for optical monitoring and dissection of microbial communities

“…As shown in previous work, this larger diameter allows for low variation in community structure and can be used as a reference during experiments. 1 The 12 columns varied by pitch, where pitch is defined as the distance between each well with respect to number of equivalent well diameters, i.e., 5 lm well with 2Â pitch would have a spacing of 10 lm between each well (Fig. 3).…”

“…A 100 ll aliquot of this cell suspension was then pipetted onto the transparent microwell arrays, following an initial addition of 500 lg ml À1 bovine serum albumin (BSA) diluted in 1Â phosphate buffered saline (PBS). 1 Excess BSA-PBS solution was dried under a stream of N 2 before adding the cell culture. Upon addition of the cell suspension, the assembly was incubated in a humidified plastic box for 1 h under standard conditions.…”

“…Following the removal of parylene and unseeded cells, an LB 2% agar layer was cast on a separate glass coverslip of the same dimensions and placed on top of the microwell arrays to provide nutrient flux to growing cells over an 18 h period, as described previously. 1 The arrays were then imaged using epifluorescence and brightfield illumination.…”

“…We have recently developed and described a microwell array platform that allows both deterministic and stochastic assembly of microbial communities for large scale monitoring and testing. 1 Here, we describe the fabrication and testing of an enhanced transparent version of that platform that retains the functionality of its predecessor, while dramatically expanding the imaging modalities that can be used to monitor the platform and improve fluidic access to the chemical microenvironment.…”

“…Previous work has shown that microwell arrays retain the ability to address both spatial organization and environmental diversity regarding nutrient exchange within microbial communities. 1,5 These platforms require limited or no connections to flow control systems and are highly compatible with conventional microscopes. Other high-throughput approaches include microfluidic droplet systems, where bacteria are isolated within a defined aqueous phase.…”