Defined spatial structure stabilizes a synthetic multispecies bacterial community

Abstract

This paper shows that for microbial communities, "fences make good neighbors." Communities of soil microorganisms perform critical functions: controlling climate, enhancing crop production, and remediation of environmental contamination. Microbial communities in the oral cavity and the gut are of high biomedical interest. Understanding and harnessing the function of these communities is difficult: artificial microbial communities in the laboratory become unstable because of "winner-takes-all" competition among species. We constructed a community of three different species of wild-type soil bacteria with syntrophic interactions using a microfluidic device to control spatial structure and chemical communication. We found that defined microscale spatial structure is both necessary and sufficient for the stable coexistence of interacting bacterial species in the synthetic community. A mathematical model describes how spatial structure can balance the competition and positive interactions within the community, even when the rates of production and consumption of nutrients by species are mismatched, by exploiting nonlinearities of these processes. These findings provide experimental and modeling evidence for a class of communities that require microscale spatial structure for stability, and these results predict that controlling spatial structure may enable harnessing the function of natural and synthetic multispecies communities in the laboratory.

Fig. 1.

A synthetic community of three bacterial species requires spatial structure to maintain stable coexistence. (A) A schematic drawing of the wild-type soil bacteria and their functions used to create a synthetic community with syntrophic interactions. (B) Graphs show the survival ratio of each species (N/N_o) as a function of time when cultured in well-mixed conditions in a test tube in nutrient-rich TSB/1771 (Left) and nutrient-poor CP (Right) media, indicating instability of the community under spatially unstructured conditions. (C) A schematic drawing of the microfluidic device used to co-culture the three species stably by imposing spatial structure with three culture wells and a communication channel.

Fig. 2.

Stability of the community in the nutrient-poor CP medium requires communication among the three species. (A) Fluorescence images of all three species in the microfluidic device at t = 0 (Top) and at t = 36 h (Bottom). Each species was cultured in an individual culture well of the microfluidic device. (B) Fluorescence images of an isolated species in the microfluidic device at t = 36 h. The same species occupied all three culture wells. Images at t = 0 were similar to those for the three species community at t = 0 (A, Top) and are not shown. Bacteria were stained with a fluorescent dye to indicate live (green) and dead (red) cells. Scale bars represent 50 μm. (C) Graphs comparing the number of live bacteria over time in devices containing all three species, each in an individual well (open squares) and in devices containing a single species in all three wells (closed triangles). Error bars represent standard error with n = 3, except for Av, 0 h (n = 4) and Bl, community, 24 h; Pc, community, 12 h; Pc, community, 36 h; and Pc, isolated species, 36 h (n = 2). P values were calculated by using two-way ANOVA.

Fig. 3.

Synthetic community coexists only at intermediate separations. (A) A schematic drawing (Left) of a mixed culture of all three species in each well of the microfluidic device and representative fluorescent images (Right) of a culture well containing all three species over time. Bacteria were stained to indicate live (green) and dead (red) cells. Scale bar represents 50 μm. (B) Graph comparing the normalized number of live cells of each species in devices with culture wells separated by four different distances. In the 0-μm separation distance, total numbers of all three species were counted. Error bars represent standard errors.

Fig. 4.

Mathematical model of a two-species syntrophic community. In all panels, the green plane represents the concentration of nutrient B at species α, [B]_α; the blue surfaces and curves represent the production rate of nutrient A; the red plane and lines represent the total rate of consumption of A; and the gray planes and lines represent the rate of consumption of A by species α only. (A) A schematic diagram showing that colony α produces nutrient A and colony β produces nutrient B, establishing gradients over the distance between the colonies L (m). The thickness of the arrows represents the continuous change of concentrations of A and B. (B) 3D rate plot of changes of consumption of A as a function of L. (C) 3D plot of changes in [B]_α as a function of L. (D–I) 3D rate plots and 2D sections of the state of the community when colonies of species α and β are separated by small (D and G), intermediate (E and H), and large (F and I) L. The steady-state concentration of nutrient A occurs where the consumption and production curves intersect, shown by the dashed white line in E and F, at the given concentration of nutrient B. (D–F) 3D rate plots from a Class II community predict a non-zero steady state only at intermediate L, indicating that the synthetic community experimentally tested here is Class II. (G–I) 2D sections of representative production and consumption curves for Class I, II, and III communities. (J and K) Steady-state concentrations of A and B and colony sizes of α and β as a function of distance parameter d; see SI Text for details.