Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium Katie Brenner, Frances H. Arnold* Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California, United States of America Abstract Microbial consortia constitute a majority of the earth’s biomass, but little is known about how these cooperating communities persist despite competition among community members. Theory suggests that non-random spatial structures contribute to the persistence of mixed communities; when particular structures form, they may provide associated community members with a growth advantage over unassociated members. If true, this has implications for the rise and persistence of multi-cellular organisms. However, this theory is difficult to study because we rarely observe initial instances of non-random physical structure in natural populations. Using two engineered strains of Escherichia coli that constitute a synthetic symbiotic microbial consortium, we fortuitously observed such spatial self-organization. This consortium forms a biofilm and, after several days, adopts a defined layered structure that is associated with two unexpected, measurable growth advantages. First, the consortium cannot successfully colonize a new, downstream environment until it self- organizes in the initial environment; in other words, the structure enhances the ability of the consortium to survive environmental disruptions. Second, when the layered structure forms in downstream environments the consortium accumulates significantly more biomass than it did in the initial environment; in other words, the structure enhances the global productivity of the consortium. We also observed that the layered structure only assembles in downstream environments that are colonized by aggregates from a previous, structured community. These results demonstrate roles for self-organization and aggregation in persistence of multi-cellular communities, and also illustrate a role for the techniques of synthetic biology in elucidating fundamental biological principles. Citation: Brenner K, Arnold FH (2011) Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium. PLoS ONE 6(2): e16791. doi:10.1371/journal.pone.0016791 Editor: Najib El-Sayed, The University of Maryland, United States of America Received August 27, 2010; Accepted January 12, 2011; Published February 9, 2011 Copyright: 2011 Brenner, Arnold. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The work was supported by the National Institutes of Health grant 1-R01-CA118486. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: frances@cheme.caltech.edu Introduction The vast majority of living biomass consists of single-celled organisms, but the existence of higher organisms demonstrates that interacting networks of cell populations can thrive despite competi- tion between them [1,2]. How nascent communities gain a growth advantage over unassociated individuals is an open question [2–9], but cell–cell interactions [10–14] and the formation of specific multi- cellular structures [15–18] are thought to contribute. Evaluating the role of physical structure in the initiation and persistence of natural consortia poses a causality dilemma [2], and de novo design of synthetic consortia that self-organize into specific structures is difficult. Thus, experimental studies of the formation and benefits of specific physical structures in mixed microbial communities are few. We describe a synthetic symbiotic microbial consortium that allows us to address some of these questions. An advantage of using synthetic, or engineered, consortia for studies of this nature is that complex communal behaviors such as symbiosis can be implemented under defined and tunable experimental control [9,19–21]. Although very simple relative to naturally-occurring microbial consortia, engineered ecosystems can nonetheless exhibit behaviors that mimic those found in nature and, because the interactions of engineered consortia can be controlled and more fully characterized, can provide insight into the development and persistence of natural communities. It is useful to study the relationship between microbial community structure and persistence in biofilm communities for three primary reasons. First, biofilm spatial structure and productivity (total biomass accumulation) can be observed and quantified as a function of time using confocal laser scanning microscopy (CLSM) [22]. Second, stable micro-communities with very different properties and behaviors can form and persist within biofilms [23–26]. Finally, cells and sub-communities that detach from a biofilm subjected to fluid flow will flow downstream and may colonize downstream environments, where composition, spatial structure, and productivity can be observed. Thus, the effects of composition and spatial structure on the productivity of a consortium can be easily quantified when it grows as a biofilm. A structure that is beneficial should increase productivity in the local environment; it might also improve colonization or productivity when the consortium moves to downstream environments. The synthetic microbial consortium was engineered to rely on biofilm formation so that these effects could be measured. Results The synthetic symbiotic consortium consists of two engineered populations of Escherichia coli which are not viable alone, but can grow and form biofilms when grown together (Fig 1A). The first PLoS ONE | www.plosone.org 1 February 2011 | Volume 6 | Issue 2 | e16791