Stochastic assembly of bacteria in microwell arrays reveals the importance of confinement in community development

Ryan H. Hansen; Andrea C. Timm; Collin M. Timm; Amber N. Bible; Jennifer L. Morrell-Falvey; Dale A. Pelletier; Michael L. Simpson; Mitchel J. Doktycz; Scott T. Retterer

doi:10.1371/journal.pone.0155080

Stochastic assembly of bacteria in microwell arrays reveals the importance of confinement in community development

Ryan H. Hansen, Andrea C. Timm, Collin M. Timm, Amber N. Bible, Jennifer L. Morrell-Falvey, Dale A. Pelletier, Michael L. Simpson, Mitchel J. Doktycz, Scott T. Retterer

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37 Scopus citations

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. Multimember 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.

Original language	English
Article number	e0155080
Journal	PLoS ONE
Volume	11
Issue number	5
DOIs	https://doi.org/10.1371/journal.pone.0155080
State	Published - May 1 2016

Funding

This work was supported in part by the Genomic Science Program, U.S. Department of Energy, Office of Science, Biological and Environmental Research, as part of the Plant Microbe Interfaces Scientific Focus Area ( http://pmi.ornl.gov ) as well as ORNL Laboratory Directed Research and Development. Microwell arrays were fabricated and characterized at the Center for Nanophase Materials Sciences User Facilities Division, Office of Basic Energy Sciences, U.S. Department of Energy. The authors would also like to thank the J. Mougous Laboratory (University of Washington, Seattle, WA) for the supply of P. aeruginosa strains used in these studies and Dr. B. Tseng Laboratory (University of Washington, Seattle, WA) for the supply of plasmids used to express GFP and mCherry in E. coli. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funders	Funder number
U.S. Department of Energy
Office of Science
Biological and Environmental Research
Laboratory Directed Research and Development

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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. Multimember 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.",

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Stochastic assembly of bacteria in microwell arrays reveals the importance of confinement in community development

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