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Abstract Bacterial biofilms, surface-attached microbial communities, grow horizontally across surfaces and vertically above them. Although a simple heuristic model for vertical growth was experimentally shown to accurately describe the behavior of diverse microbial species, the biophysical implications and theoretical basis for this empirical model were unclear. Here, we demonstrate that this heuristic model emerges naturally from fundamental principles of active fluid dynamics. By analytically deriving solutions for an active fluid model of vertical biofilm growth, we show that the governing equations reduce to the same form as the empirical model in both early- and late-stage growth regimes. Our analysis reveals that cell death and decay rates likely play key roles in determining the characteristic parameters of vertical growth. The active fluid model produces a single, simple equation governing growth at all heights that is surprisingly simpler than the heuristic model. With this theoretical basis, we explain why the vertical growth rate reaches a maximum at a height greater than the previously identified characteristic length scale. These results provide a theoretical foundation for a simple mathematical model of vertical growth, enabling deeper understanding of how biological and biophysical factors interact during biofilm development. 

The ecological and evolutionary success of multicellular lineages stems substantially from their increased size relative to unicellular ancestors. However, large size poses biophysical challenges, especially regarding nutrient transport: These constraints are typically overcome through multicellular innovations. Here, we show that an emergent biophysical mechanism—spontaneous fluid flows arising from metabolically generated density gradients—can alleviate constraints on nutrient transport, enabling exponential growth in nascent multicellular clusters of yeast lacking any multicellular adaptations for nutrient transport or fluid flow. Beyond a threshold size, the metabolic activity of experimentally evolved snowflake yeast clusters drives large-scale fluid flows that transport nutrients throughout the cluster at speeds comparable to those generated by ciliary actuation in extant multicellular organisms. These flows support exponential growth at macroscopic sizes that theory predicts should be diffusion limited. This demonstrates how simple physical mechanisms can act as a “biophysical scaffold” to support the evolution of multicellularity by opening up phenotypic possibilities before genetically encoded innovations. 

Oxygen availability is a key factor in the evolution of multicellularity, as larger and more sophisticated organisms often require mechanisms allowing efficient oxygen delivery to their tissues. One such mechanism is the presence of oxygen-binding proteins, such as globins and hemerythrins, which arose in the ancestor of bilaterian animals. Despite their importance, the precise mechanisms by which oxygen-binding proteins influenced the early stages of multicellular evolution under varying environmental oxygen levels are not yet clear. We address this knowledge gap by heterologously expressing the oxygen-binding proteins myoglobin and myohemerythrin in snowflake yeast, a model system of simple, undifferentiated multicellularity. These proteins increased the depth and rate of oxygen diffusion, increasing the fitness of snowflake yeast growing aerobically. Experiments show that, paradoxically, oxygen-binding proteins confer a greater fitness benefit for larger organisms when O₂is least limiting. We show via biophysical modeling that this is because facilitated diffusion is more efficient when oxygen is abundant, transporting a greater quantity of O₂which can be used for metabolism. By alleviating anatomical diffusion limitations to oxygen consumption, the evolution of oxygen-binding proteins in the oxygen-rich Neoproterozoic may have been a key breakthrough enabling the evolution of increasingly large, complex multicellular metazoan lineages. 

The study of interfacial roughening is common in physics, from epitaxial growth in the lab to pio-neering mathematical descriptions of universality in models of growth processes. These studies led to the identification of a series of general principles. Typically, stochastic growth produces an interface that becomes rougher as the deposit grows larger; this roughening can only be counteracted by mechanisms that act on the top of deposit, such as surface tension or surface diffusion. However, even when relaxation mechanisms are present, interfaces that continue to grow stochastically continue to change; new peaks and troughs emerge and disappear as stochastic growth produces a constantly changing, dynamic interface. These universal phenomena have been observed for bacterial colonies in a variety of contexts. However, previous studies have not characterized the interfacial phenomena at the top surface of a colony, i.e., the colony-air interface, when activity is only present at the bottom surface, i.e., the colony-solid interface, where nutrients are available, over long times. As traditional interfacial roughening models primarily focus on activity occurring at the top surface it is unclear what phenomena to expect over long times. Here, we use white light interferometry to study the roughening of bacterial biofilms, from many different species. We find that these colonies are remarkably smooth, suggesting that a mechanism of interfacial relaxation is at play. However, colonies remain remarkably smooth even after growing large. We discover that topographic fluctuations “freeze” in place, despite the fact that growth continues for hundreds of microns more. With simple simulations, we show that this emergent freezing is due to the dampening of fluctuations from cell growth by the cells between the growing zone and the surface. We find that the displacement field caused by a single perturbation decays exponentially, with a decay length ofδL. In line with that observation we also show that the topography ceases to change when perturbations are a distanceδLaway from the surface. Thus, over-damped systems in which activity occurs at the bottom surface represent a distinct class of interfacial growth phenomena, capable of producing frozen topographies and remarkably smooth surfaces from spatially and temporally stochastic growth. 

The ecological and evolutionary success of multicellular lineages is due in no small part to their increased size relative to unicellular ancestors. However, large size also poses biophysical challenges, especially regarding the transport of nutrients to all cells; these constraints are typically overcome through multicellular innovations (e.g., a circulatory system). Here we show that an emergent biophysical mechanism — spontaneous fluid flows arising from metabolically-generated density gradients — can alleviate constraints on nutrient transport, enabling exponential growth in nascent multicellular clusters of yeast lacking any multicellular adaptations for nutrient transport or fluid flow. Surprisingly, beyond a threshold size, the metabolic activity of experimentally-evolved snowflake yeast clusters drives large-scale fluid flows that transport nutrients throughout the cluster at speeds comparable to those generated by the cilia of extant multicellular organisms. These flows support exponential growth at macroscopic sizes that theory predicts should be diffusion limited. This work demonstrates how simple physical mechanisms can act as a ‘biophysical scaffold’ to support the evolution of multicellularity by opening up phenotypic possibilities prior to genetically-encoded innovations. More broadly, our findings highlight how cooption of conserved physical processes is a crucial but underappreciated facet of evolutionary innovation across scales. 

Surface attached communities of microbes grow in a wide variety of environments. Often, the size of these microbial community is constrained by their physical surroundings. However, little is known about how size constraints of a colony impact the outcome of microbial competitions. Here, we use individual-based models to simulate contact killing between two bacterial strains with different killing rates in a wide range of community sizes. We found that community size has a substantial impact on outcomes; in fact, in some competitions the identity of the most fit strain differs in large and small environments. Specifically, when at a numerical disadvantage, the strain with the slow killing rate is more successful in smaller environments than in large environments. The improved performance in small spaces comes from finite size effects; stochastic fluctuations in the initial relative abundance of each strain in small environments lead to dramatically different outcomes. However, when the slow killing strain has a numerical advantage, it performs better in large spaces than in small spaces, where stochastic fluctuations now aid the fast killing strain in small communities. Finally, we experimentally validate these results by confining contact killing strains ofVibrio choleraein transmission electron microscopy grids. The outcomes of these experiments are consistent with our simulations. When rare, the slow killing strain does better in small environments; when common, the slow killing strain does better in large environments. Together, this work demonstrates that finite size effects can substantially modify antagonistic competitions, suggesting that colony size may, at least in part, subvert the microbial arms race. 

The evolution of multicellular life spurred evolutionary radiations, fundamentally changing many of Earth’s ecosystems. Yet little is known about how early steps in the evolution of multicellularity affect eco-evolutionary dynamics. Through long-term experimental evolution, we observed niche partitioning and the adaptive divergence of two specialized lineages from a single multicellular ancestor. Over 715 daily transfers, snowflake yeast were subjected to selection for rapid growth, followed by selection favouring larger group size. Small and large cluster-forming lineages evolved from a monomorphic ancestor, coexisting for over ~4,300 generations, specializing on divergent aspects of a trade-off between growth rate and survival. Through modelling and experimentation, we demonstrate that coexistence is maintained by a trade-off between organismal size and competitiveness for dissolved oxygen. Taken together, this work shows how the evolution of a new level of biological individuality can rapidly drive adaptive diversification and the expansion of a nascent multicellular niche, one of the most historically impactful emergent properties of this evolutionary transition. 

Bacteria often attach to surfaces and grow densely-packed communities called biofilms. As biofilms grow, they expand across the surface, increasing their surface area and access to nutrients. Thus, the overall growth rate of a biofilm is directly dependent on its “range expansion” rate. One factor that limits the range expansion rate is vertical growth; at the biofilm edge there is a direct trade-off between horizontal and vertical growth—the more a biofilm grows up, the less it can grow out. Thus, the balance of horizontal and vertical growth impacts the range expansion rate and, crucially, the overall biofilm growth rate. However, the biophysical connection between horizontal and vertical growth remains poorly understood, due in large part to difficulty in resolving biofilm shape with sufficient spatial and temporal resolution from small length scales to macroscopic sizes. Here, we experimentally show that the horizontal expansion rate of bacterial colonies is controlled by the contact angle at the biofilm edge. Using white light interferometry, we measure the three-dimensional surface morphology of growing colonies, and find that small colonies are surprisingly well-described as spherical caps. At later times, nutrient diffusion and uptake prevent the tall colony center from growing exponentially. However, the colony edge always has a region short enough to grow exponentially; the size and shape of this region, characterized by its contact angle, along with cellular doubling time, determines the range expansion rate. We found that the geometry of the exponentially growing biofilm edge is well-described as a spherical-cap-napkin-ring, i.e., a spherical cap with a cylindrical hole in its center (where the biofilm is too tall to grow exponentially). We derive an exact expression for the spherical-cap-napkin-ring-based range expansion rate; further, to first order, the expansion rate only depends on the colony contact angle, the thickness of the exponentially growing region, and the cellular doubling time. We experimentally validate both of these expressions. In line with our theoretical predictions, we find that biofilms with long cellular doubling times and small contact angles do in fact grow faster than biofilms with short cellular doubling times and large contact angles. Accordingly, sensitivity analysis shows that biofilm growth rates are more sensitive to their contact angles than to their cellular growth rates. Thus, to understand the fitness of a growing biofilm, one must account for its shape, not just its cellular doubling time.