![]() Bottom image was acquired near the glass surface, top image is 5 µm above). mesenteroides was still present 5 µm above (×20 magnification, scale bar represents 20 µm. lactis was only present at the bottom in close proximity to the glass surface, while L. mesenteroides and sfGFP(Bs)-expressing L. D Visualization of DAPI-stained coculture of L. lactis incubated in mono culture and in co-culture with L. B, C Representative CLSM images of sfGFP(Bs)-expressing L. The x-axis shows the log10 transformed biovolume. Quantification of biovolume was performed using RCon3D and all replicates were imaged in a fixed volume of space (X:Y:Z 319.45 µm:319.45 µm:12 µm). mesenteroides on a glass surface compared to those of the single culture (P adj = 0.023, Welch Two Sample t test, N = 3). lactis harboring pNAMA_P32-sfgfp(Bs)-CmR were lower when co-cultivated with L. npj Biofilms and Microbiomes (2022) 8:59 CLSM quantification of Lactococcus lactis abundance and localization on glass slides. Our study underlines the power of ecological thinking-namely, the importance of structured environments for coexistence-to facilitate cultivation, evolution, and adaptation of industrially important bacterial communities. Therefore, both co-culture and the biofilm model were conditional for these high-yield strains to evolve. Importantly, we also show that these high-yield biofilm strains did not evolve when each species was passaged in monoculture in the biofilm model. Moreover, we show that the co-culture drove evolution of new high-yield variants, which compared to the ancestor grew more slowly, yielded more cells and had enhanced capability of biofilm formation. By contrast, when we co-cultured the two species as biofilms on beads, a stable coexistence was observed in all lineages studied for over 100 generations. As commonly occurs, passaging of these two species together in a liquid culture model led to the loss of one species in 6 of 20 lineages (30%). Here, we explored the ability of cultivation in a structured environment to facilitate coexistence, evolution, and adaptation in an industrially important community: Lactococcus lactis and Leuconostoc mesenteroides frequently used as dairy starter cultures. Ecological theory predicts that a solution to this problem will be to grow species in structured environments, which reduces the likelihood of competitive exclusion. This diversity presents a significant challenge as co-cultivation of multiple bacterial species frequently leads to species being outcompeted and, with this, the possibility to manipulate, evolve and improve bacterial communities is lost. The majority of ecological, industrial and medical impacts of bacteria result from diverse communities containing multiple species. ![]()
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