Microbial competition in porous environments can PNAS PLUS select against rapid biofilm growth Katharine Z. Coytea,b,c,d, Herve´ Tabuteaue, Eamonn A. Gaffneyb, Kevin R. Fostera,c,1, and William M. Durhama,f,1 aDepartment of Zoology, University of Oxford, Oxford OX1 3PS, United Kingdom; bWolfson Centre for Mathematical Biology, University of Oxford, Oxford OX2 6GG, United Kingdom; cOxford Centre for Integrative Systems Biology, University of Oxford, Oxford OX1 3QU, United Kingdom; dComputational Biology, Memorial Sloan Kettering Cancer Center, New York, NY 10065; eInstitut de Physique de Rennes, Universite´ de Rennes 1, 35042 Rennes, France; and fDepartment of Physics and Astronomy, University of Sheffield, Sheffield S3 7RH, United Kingdom Edited by Howard A. Stone, Princeton University, Princeton, NJ, and approved November 1, 2016 (received for review December 21, 2015) Microbes often live in dense communities called biofilms, where constraints. In porous environments, space is much more limited, competition between strains and species is fundamental to both and biofilm growth tends to attenuate the fluid flow that supplies evolution and community function. Although biofilms are com- cells with nutrients and facilitates dispersal. monly found in soil-like porous environments, the study of micro- Biofilms typically reduce the flow through porous environ- bial interactions has largely focused on biofilms growing on flat, ments by orders of magnitude at the Darcy scale (22), a macro- planar surfaces. Here, we use microfluidic experiments, mechanis- scopic scale that measures the flow averaged over many pore tic models, and game theory to study how porous media hydro- spaces. Harnessing this effect, biofilms can be used to limit dynamics can mediate competition between bacterial genotypes. the transport of pollutants that have leaked into groundwater Our experiments reveal a fundamental challenge faced by micro- aquifers and to facilitate the extraction of petroleum from recal- bial strains that live in porous environments: cells that rapidly citrant regions of reservoirs (23, 24). However, biofilm-induced form biofilms tend to block their access to fluid flow and redi- clogging also generates unwanted effects: for example, it severely rect resources to competitors. To understand how these dynam- limits the efficiency of porous filtration systems (25) and curtails ics influence the evolution of bacterial growth rates, we couple the rate at which water infiltrates into aquifers (26), exacerbat- a model of flow–biofilm interaction with a game theory anal- ing droughts. Due to its importance, the attenuation of flow by ysis. This investigation revealed that hydrodynamic interactions biofilms has long been studied at the Darcy scale (27, 28), and between competing genotypes give rise to an evolutionarily sta- more recent works have sought to resolve how this process, in ble growth rate that stands in stark contrast with that observed in turn, is mediated by biofilm–hydrodynamic interactions at the typical laboratory experiments: cells within a biofilm can outcom- microscopic pore scale (29–31). However, it is largely unknown pete other genotypes by growing more slowly. Our work reveals how these interactions influence the ecology and evolution of the that hydrodynamics can profoundly affect how bacteria com- bacteria themselves. Here, we combine experiments and mod- pete and evolve in porous environments, the habitat where most els to show that porous media hydrodynamics can dramatically bacteria live. affect the principles of bacterial competition and evolution. Results bacterial evolution j porous media flow j clogging j game theory j A Conceptual Model to Study Hydrodynamic Interactions Between adaptive dynamics Competing Biofilms. Bacteria within biofilms tend to form patches of genetically identical cells, even when the cells from which they odern microbiology relies on growing cells in liquid cul- tures and agar plates. Although these conditions offer high M Significance throughput and repeatability, they lack the complex physical and chemical landscapes that microbes experience in their natural environments. This environmental heterogeneity is increasingly The overwhelming majority of bacteria live in porous envi- recognized to exert a powerful influence on microbial ecology ronments, like soil, aquifers, and sediments, where they facil- across a wide diversity of habitats, ranging from the ocean to the itate many important processes. Despite their importance, human gut (1–4). Although advances in sequencing technology we understand little about how these complex environments now allow us to resolve how the genetic composition of microbial shape the composition of the microbial communities that communities changes in response to environmental conditions live within them. Here, we combine two diverse bodies of (5, 6), we often lack a mechanistic understanding of the underly- theory—fluid dynamics and game theory—to shed light on ing processes. Novel empirical approaches, which simulate the how bacteria evolve in these habitats. We show that bac- conditions found in realistic microbial habitats, are needed to teria in porous environments face a fundamental dilemma: understand the strategies that cells use to gain an advantage over they rely on flow for nutrients and dispersal; however, as cells their competitors (7). grow, they tend to reduce their access to flow. A fast growing strain can, therefore, choke off its own nutrient supply, divert- The overwhelming majority of bacteria live in porous environ- ing it instead to competitors. In contrast with classical the- ments between the particles that compose soil, aquifers, and sed- ory, our results suggest that cells within a biofilm can obtain iments, and cumulatively comprise roughly half of the carbon a competitive advantage by growing more slowly. EVOLUTION within living organisms globally (8). Cells in porous environments typically reside in surface attached structures known as biofilms Author contributions: K.Z.C., H.T., and W.M.D. designed and performed the microfluidic (9), in which diverse bacterial genotypes live under intense com- experiments; K.Z.C., E.A.G., K.R.F., and W.M.D. developed the mathematical models; and petition for limited resources (10, 11). Recent efforts have iden- K.Z.C., E.A.G., K.R.F., and W.M.D. wrote the paper. tified specialized mechanisms that cells use to gain advantage The authors declare no conflict of interest. over competing genotypes in biofilms, ranging from the secretion This article is a PNAS Direct Submission. of toxins to polymer production and metabolic regulation (12– 1To whom correspondence may be addressed. Email: [email protected] or 18). Whereas genotypic competition is most frequently studied in w.m.durham@sheffield.ac.uk. PHYSICS biofilms growing on simple flat surfaces (19–21), biofilms growing This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in the interstitial spaces within porous structures face additional 1073/pnas.1525228113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1525228113 PNAS j Published online December 22, 2016 j E161–E170 Downloaded by guest on September 25, 2021 are founded are initially mixed. This genotypic patchiness occurs genotypes were inoculated separately in either arm of the device, because in situ growth, combined with the low mobility of cells each of which represents a pore (Fig. 2 A and B). One of the within biofilms, means that clone mates tend to remain in close pores was irrigated with media mixed with dye, whereas the other proximity to one another (32, 33). Moreover, genotypic patch- was irrigated with clear media, which allowed us to measure the iness in biofilms is enhanced by population bottlenecks, which relative proportion of flow passing through each pore by tracking occur more frequently in nutrient-limited conditions, and when the dye interface downstream of their juncture (SI Text and Fig. biofilms are initiated from a sparse distribution of attached cells S1). Control experiments showed that neither the dye nor the (34–36). Based on these observations, we focus here on the com- fluorescent proteins used to differentially label the strains had petition between localized biofilm “patches” that each comprise an appreciable effect on biofilm formation (Fig. S2). a single genotype and assume that competing patches occupy dif- In porous environments biofilm growth is opposed by flow- ferent pore spaces. induced detachment, which reduces the thickness of biofilms by To investigate how biofilm growth influences the flow through shearing away cells from its surface (40–42). To simulate differ- a porous environment, we calculated the Stokes flow through a ent ambient flow conditions in our experiment, and thus the rela- representative network of pore spaces that is driven by a differ- tive amount of detachment, we applied a total flow rate of either −1 −1 ence in pressure at the boundaries (Fig. 1A and Materials and QT = 0:1 mL h or QT = 2 mL h . In the low-flow treat- Methods). The addition of a small impermeable biofilm patch ment, the rapidly expanding wild-type biofilm increased its pore’s sharply reduces the flow through the pore in which the biofilm hydrodynamic resistance and diverted flow to the neighboring resides, while concurrently increasing the flow through neigh- pore space containing the ∆rpoS biofilm. The reduction in flow boring pores (Fig. 1 B and C). Although the magnitude of this experienced by the wild-type biofilm further reduces its detach- flow diversion depends on the specific geometry of the pore ment, driving a positive-feedback loop that ultimately ends with space, this simulation shows that as a biofilm patch proliferates, the ∆rpoS biofilm capturing nearly all of the flow (Fig. 2 A, C, it tends to decrease its access to flow, while increasing the flow to and D; see SI Text for details). In contrast, under the high-flow patches of biofilm that reside along other flow paths. This diver- treatment, the flow-induced detachment is increased so that both sion of flow introduces a way in which biofilms can interact: geno- genotypes form much thinner biofilms (Fig.