Session Q05: Biofilms

Surface tension regulates the morphological evolution of a growing bacterial colony at an air-solid interface

There are several medically and industrially important contexts in which bacteria grow on surfaces to form biofilms at an interface between air and porous media. However, the dynamics associated with bacterial growth from a single cell and its evolution into a 3D biofilm at such interfaces remain largely unknown. Here, we study the early morphological evolution of an E. Coli colony growing on the surface of an agarose gel (open to air) as it grows from a single cell into a colony of over ~10,000 cells. We use laser scanning microscopy to measure the 3D shape of a growing biofilm and vary agarose gel concentration to systematically control for water availability around the cells. Starting from a single cell, we observe an initial growth phase where E. coli cells on the agarose surface primarily grow and divide in-plane in two-dimensions. Thereafter, we observe a distinctive out-of-plane morphological evolution, which we hypothesize is initiated by a mechanical instability arising from an interplay among growth-associated mechanical stresses, cell-cell and cell-substrate adhesion, and surface tension. Our observations suggest a model in which surface tension forces are important in regulating the overall shape and evolution of bacterial colonies growing at air-solid interfaces.   

Study of 3D bacterial motion and biofilm formation by digital holographic microscopy

Digital Holographic Microscopy (DHM) was applied to study the 3D bacterial motion and biofilm formation.The bacterial sample was placed in a closed chamber with a thickness of 180 µm and the bacterial motion recorded at 50 frames per second continuously for 40 s at every 1 hour. The experiment began at a low bacterial concentration of 500 cells/mm³ and ran for a duration of 4 hours by which the concentration increased to 6000 cells/mm³. The holograms were post-processed to generate 3D trajectories of bacteria. Two strains of Shewanella sp. were studied, one showing preferential growth near the surface (i.e., creating biofilm), while the other exhibiting similar growth rates throughout the chamber (i.e., no detectable biofilm formation). We analyzed and compared the velocity and mean-square-displacement (MSD) of the two strains. We found that in the near-wall region, both bacterial strains have small velocity and small MSD. While in the bulk region, the strain creating biofilm has a much large velocity (50 µm/s) and a large MSD compared to the one showing no preference growth. The large velocity may indicate that the bacteria are actively searching for solid surface. Our result suggests that biofilm formation is related to the bacterial swimming behavior in the bulk region.   

Biofilm viscoelasticity and nutrient source location control biofilm growth rate, migration rate, and morphology in shear flow

Our numerical model to simulate the growth and deformation of a viscoelastic biofilm in shear flow under different nutrient conditions uses the Immersed Boundary Method with viscoelastic parameters determined a priori from measurements reported in the literature. Biofilm growth occurs at the biofilm-fluid interface by a stochastic rule that depends on the local nutrient concentration. We compare the growth, migration, and morphology of viscoelastic biofilms with a common relaxation time of 18 min over the range of elastic moduli 10-1000 Pa in different nearby nutrient source configurations. Our results agree with the biophysical principle that biofilms can adapt to their mechanical and chemical environment by modulating their viscoelastic properties. Furthermore, by comparing the behavior of a purely elastic biofilm to a viscoelastic biofilm with the same elastic modulus, we find that the elastic biofilm underestimates growth rates and downstream migration rates if the nutrient source is downstream, and it overestimates growth rates and upstream migration rates if the nutrient source is upstream. Future modeling can use our comparison to identify errors that can occur by simulating biofilms as purely elastic structures.   

Biofilm formation behind a backward facing step

Flow properties, such as wall shear stress, and nutrient availability have been shown to influence the formation of biofilm. Disentangling wall shear stress and nutrient transport is still a challenge. Prior studies have mostly focused on straight channels or pipes where homogeneous conditions are encountered at the walls. We aim to connect the study of biofilm formation with the case of a backward-facing step type flow. Within the recirculation area, nutrient transport is dominated by diffusion processes, whereas outside of it advection dominates. The influence of spatially varying shear stress and nutrient availability on biofilm formation is investigated. The morphology of a pseudomonas fluorescens biofilm forming behind the backward-facing step is measured using optical coherence tomography. We show that early biofilm growth is concentrated in areas with low velocities that are still dominated by advective nutrient transport as well as the stagnation point.   

Sinking behavior of biofilm-covered microplastics with irregular shape: computer aided simulation and experimental validation

We use computational fluid dynamics to investigate the sinking behavior of biofilm-covered microplastics (BMPs) with realistic shape in a water column. The structure of individual BMPs is either acquired from imaging experiments [1], or generated through computer simulation [2]. The Navier-Stokes-Brinkman formulation is used for the flow pattern around the BMP and within the poroelastic biofilm. The gravity-driven motion of the BMP is simulated with the immersed boundary method. Simulations are parameterized by the Reynolds and Darcy numbers that capture the combined effects of particle size, excess density, seawater viscosity and biofilm permeability. At steady-state sinking, high-resolution finite element analysis provides the distribution of elastic stresses within the biofilm and the locations of maximum likelihood for detachment. The computed terminal velocity is compared with measurements from particle tracking velocimetry experiments [1], across a wide range of aggregate geometry and biological fraction.

[1] Nguyen TH, Tang FHM, Maggi F (2020). Sinking of microbial-associated microplastics in natural waters. PLoS ONE 15(2): e0228209.

[2] Kapellos, G.E., Alexiou, T.S. and Pavlou, S (2015). Fluid-biofilm interactions in porous media, Elsevier, pp. 207-238.   

The potential of bio-inspired structures on anti-biofouling

Biofouling is the accumulation of biological organisms on wet surfaces. It particularly affects marine transportation by increasing skin drag and may produce local flow separation, increasing fuel costs, schedule, and maintenance breaks. Drag-reducing bioinspired structures (Humberto et al. PNAS 2018) may control biofouling by increasing local near-wall turbulence with unsteady blowing and suction mechanisms. We explored experimentally this phenomenon in terms of its ability to prevent the formation of a slime layer. We used sodium alginate solution to mimic the secretions of marine organisms. A dye test was used to examine the formation of the slime layer with and without surface coating under turbulence and laminar flow regimes. We will quantitatively discuss the flow interaction in the bio-inspired structures and the slime from micro-scope and PIV techniques.   

Effects of surface energy landscape on shear resistant biofilm structures in high shear flows

Biofilm consisting of structured bacterial communities protected cells from environmental insults such as antibiotics, biocides, and mechanical abrasions.  Recent studies on near surface bacterial motility and biofilm responses to flow shear leads to hypothesis that substrate landscape (e.g. surface hydrophobicity, roughness, and chemistry) and hydrodynamic conditions (e.g. flow and shear) substantially affect the fundamental formation processes and cause the film to evolve to diametrically different mature biofilms. In this study, we apply our newly developed Ecology-on-a-chip (eChip) microfluidic microcosm platform and surface-patterning technique with OTS self-assembled monolayer micro-patches to study the role of surficial energy landscape and flow shear on 3D biofilm structures. Pseudomonas biofilms are be formed in-situ in eChip platform with a SMA patterned bottom surface. The formation processes (e.g. attachment, proliferation, dispersal, regrowth) and the evolution of 3D film structures are measured in the real-time as well as their capability of resisting shear will also be assessed and quantified. Results show that direct correlations of length and time scale between energy landscape and near wall flow.   

In-situ study of shear resistant (SR-) biofilm formation in a mesoscale turbulent channel facility

Biofouling of ship hulls is a ubiquitous phenomenon and causes substantial maintenance costs each year. While real-world flow near a ship hull is highly turbulent, past laboratory studies focus largely on understanding biofilm formation mechanisms and identification of phenotypical responses by microbes to a wall under low and laminar flow shear. Recent findings showing biofilm formed over a liquid-liquid interface under large flow shear and capable of resisting shear erosion have reignited interest in investigating shear resistant (SR-) biofilm. To provide new insight on SR-biofilm formation at scales relevant to real-world applications, a mesoscale flow facility enabling the study of in-situ biofilm formation at high turbulent flow shear has been developed. To measure biofilm formation and feedbacks to near-film flow, the facility is instrumented for simultaneous measurements of evolving biofilm structural morphology and the flow field via coupled particle image velocimetry (PIV) and planar laser-induced fluorescence systems (PLIF). Flow conditions for testing are established via PIV measurements conducted prior to biofilm formation. In addition, experiments testing for in-situ biofilm formation from natural microbial assemblage collected at Corpus Christi Bay are performed.