Self-organization, layered structure, and aggregation enhance persistence of a synthetic biofilm consortium

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.

Figure 1. Design and initial characterization of the biofilm-forming consortium.

(A) The synthetic symbiotic consortium. The blue population cannot synthesize diaminopimelate or lysine; when cultured without lysine or diaminopimelate, this population forms only a scant biofilm. The yellow population cannot form biofilms alone but is otherwise healthy. It synthesizes C4HSL, which diffuses freely and activates production of diaminopimelate in the blue population. Yellow cells become bound within the biofilm formed by the blue population and rescue growth. Together, the two populations form viable biofilms that persist. (B) The symbiotic consortium functions as designed. The blue population control forms a biofilm which eventually dies (blue bars), and the yellow population accumulates very little biomass (insignificant, not shown). When the yellow and blue populations are inoculated into a flow chamber in a 50/50 mixture, more biomass accumulates than in either control (solid yellow areas, total yellow biomass in the biofilm; solid blue areas, total blue biomass in the biofilm; the sum of blue and yellow areas is the overall total biomass in the biofilm at each time point; all errors are s.d.). All biofilm measurements were derived from images quantified with COMSTAT .

Figure 2. The consortium adopts a specific, layered structure which is associated with a growth advantage.

(A) After 80 hours, the blue population remains primarily near the substrate while the yellow population forms clumps attached to the blue population, as shown in this cross-sectional projection taken at 1/3 the total height of the biofilm. (B) After 80 hours of growth, the yellow population begins to exhibit a consistently larger biomass median (yellow lines, throughout figure) than the blue population (blue lines, throughout figure), revealing that the yellow population grows further from the substrate while the blue population remains close to the substrate. (The biomass median indicates the average distance from the substrate at which cells of a given population are found.) Gray bars, plotted against the right-hand axis, indicate total biomass accumulation for the entire consortium at each time-point throughout the figure (errors throughout figure are s.d.). (C) Maximum total biomass accumulated by the downstream biofilm is double that in the initial biofilm (compare gray bars in [B] and [C]). Additionally, the downstream biofilm assumes the layered structure more quickly: the biomass medians reveal structure after 24 hours of growth. (D) When aggregates are disrupted prior to transfer, leaving all else constant, this treated effluent can form biofilms, but they never exhibit the layered structure or growth advantage. (E) The layered structure is recovered when the sorted blue-and-yellow aggregate fraction forms downstream biofilms. In fact, the consortium starts with this structure, exhibiting it by 24 hours after inoculation. Further, maximum total biomass accumulation is more than double the highest amount observed in the predecessor (illustrated in [D]), suggesting recovery of the growth advantage. (F) The single-cell fraction consists of more than 99% yellow cells, and thus neither the blue population nor the layered structure nor any growth advantage is evident in the downstream biofilm it forms. This biofilm accumulates less biomass than the biofilm formed by the aggregate fraction (compare to grey bars in [E]). (G) Here, effluent is taken from the treated biofilms, which are less productive and do not exhibit structure (illustrated in [D]). Although this effluent is left untreated, it forms initially dense, monomorphic, and primarily yellow downstream biofilms that do not exhibit layered structure. These biofilms consistently lose biomass.