Ecological succession in long-term experimentally evolved biofilms produces synergistic communities

Abstract

Many biofilm populations are known for their exceptional biodiversity, but the relative contributions of the forces that could produce this diversity are poorly understood. This uncertainty grows in the old, well-established communities found on many natural surfaces and in long-term, chronic infections. If the prevailing interactions among species within biofilms are positive, productivity should increase with diversity, but if they tend towards competition or antagonism, productivity should decrease. Here, we describe the parallel evolution of synergistic communities derived from a clone of Burkholderia cenocepacia during ~1500 generations of biofilm selection. This long-term evolution was enabled by a new experimental method that selects for daily cycles of colonization, biofilm assembly and dispersal. Each of the six replicate biofilm populations underwent a common pattern of adaptive morphological diversification, in which three ecologically distinct morphotypes arose in the same order of succession and persisted. In two focal populations, mixed communities were more productive than any monoculture and each variant benefited from the mixture. These gains in output resulted from asymmetrical cross-feeding between ecotypes and the expansion and partitioning of biofilm space that constructed new niches. Therefore, even in the absence of starting genetic variation, prolonged selection for surface colonization generates a dynamic of ecological succession that enhances productivity.

Figure 1

Model of long-term experimental evolution in biofilms. Six replicate populations founded with a single clone of B. cenocepacia HI2424 were propagated on 7 mm polystyrene beads, suspended in 5 ml M9+1  galactose in 15 × 180 mm test tubes on a rollerdrum at 50 r.p.m., and were sterilely transferred to new media every 24 h. Each bead population was required to colonize a new oppositely marked bead each day. P populations were serially transferred via 1:100 dilutions of planktonic cells grown in 5 ml M9+1  galactose for 24 h (not shown).

Figure 2

Adaptive diversification within Burkholderia biofilms. Colony morphologies (first row) and biofilm phenotypes (second row, growth on tube walls) of evolved variants following ∼1500 generations of biofilm selection, their timing of detection in population B1 and their associated phenotypes. All six biofilm-evolved populations produced morphologically similar types (studded=S, ruffled spreader=R, wrinkly=W; Supplementary Figure S2) that were detected during the same intervals. Morphotypes differ in their fitness, colonization patterns, growth rates and biofilm production, when grown in monoculture. Relative fitness is colonization efficiency of evolved morphotypes relative to the ancestor. Planktonic doubling time (increasing values being disadvantageous) and biofilm production were measured using standard techniques. 95% confidence intervals of each measurement are in parentheses. NA, not applicable.

Figure 3

Productivity of S, R and W morphotypes isolated from evolved biofilm populations B1 and B2 grown in monoculture and in mixed communities. Expected productivity was calculated as the product of the proportion of each morphotype in the founding population and its yield (CFU per ml) in monoculture (Loreau and Hector, 2001). Observed productivity is the total yield of the mixed community in the experimental environment. Error bars are 95% CI based on four replicates.

Figure 4

Contributions of cross-feeding and structure to positive effects of biodiversity. Morphotypes from population B1 were grown either in monoculture or in mixed communities in environments containing no bead (cross-feeding only), one bead (cross-feeding + structure) or two beads (cross-feeding+2 × structure). (a) Productivity (CFU per ml) of monocultures and mixed communities; error bars are 95% CI based on four replicates. (b) Relative effect of biodiversity (observed/expected productivity) for each morphotype in the mixed communities (symbols in legend) and the overall effect on the mixed community itself (dashed line); expected yield was calculated assuming additivity (Loreau and Hector, 2001).

Figure 5

Absolute and relative effects of cross-feeding in pairwise interactions of supernatant producers and consumers. Isolates from population B1 and the ancestor (WT) were grown in the cell-free supernatant produced by themselves and each other type. (a) Growth (measured as the area under the curve of OD600 over 24 h, ±95% CI, n=5) of each morphotype in supernatants produced by fellow community morphotypes. (b) Cross-feeding interactions between morphotypes. Numerical values indicate fold-increase in supernatant growth relative to growth in unconditioned medium. (c) Effects of growth by each morphotype in the supernatant of the mixed community of population B1, calculated as in b.

Figure 6

Confocal scanning laser microscopy of the evolved biofilm architecture. The biofilm produced by population B1 after 24 h on a polystyrene slide was rendered in three dimensions and imaged through three separate filters, a, b, and c, as follows. The entire biofilm was stained with TOPRO-3 (Invitrogen) and is projected in blue, the W morphotype carries pSPR and fluoresces red, and the R morphotype carries pSPY and is projected in green. Each morphotype inhabits a different region, and S builds clusters atop the R (indicated by yellow) and especially the W morphotype (indicated by purple).