James N. Wilking

Liquid transport facilitated by channels in Bacillus subtilis biofilms

Many bacteria on earth exist in surface-attached communities known as biofilms. These films are responsible for manifold problems, including hospital-acquired infections and biofouling, but they can also be beneficial. Biofilm growth depends on the transport of nutrients and waste, for which diffusion is thought to be the main source of transport. However, diffusion is ineffective for transport over large distances and thus should limit growth. Nevertheless, biofilms can grow to be very large. Here we report the presence of a remarkable network of well-defined channels that form in wild-type Bacillus subtilis biofilms and provide a system for enhanced transport. We observe that these channels have high permeability to liquid flow and facilitate the transport of liquid through the biofilm. In addition, we find that spatial variations in evaporative flux from the surface of these biofilms provide a driving force for the flow of liquid in the channels. These channels offer a remarkably simple system for liquid transport, and their discovery provides insight into the physiology and growth of biofilms.

Osmotic spreading of Bacillus subtilis biofilms driven by an extracellular matrix

Bacterial biofilms are organized communities of cells living in association with surfaces. The hallmark of biofilm formation is the secretion of a polymeric matrix rich in sugars and proteins in the extracellular space. In Bacillus subtilis, secretion of the exopolysaccharide (EPS) component of the extracellular matrix is genetically coupled to the inhibition of flagella-mediated motility. The onset of this switch results in slow expansion of the biofilm on a substrate. Different strains have radically different capabilities in surface colonization: Flagella-null strains spread at the same rate as wild type, while both are dramatically faster than EPS mutants. Multiple functions have been attributed to the EPS, but none of these provides a physical mechanism for generating spreading. We propose that the secretion of EPS drives surface motility by generating osmotic pressure gradients in the extracellular space. A simple mathematical model based on the physics of polymer solutions shows quantitative agreement with experimental measurements of biofilm growth, thickening, and spreading. We discuss the implications of this osmotically driven type of surface motility for nutrient uptake that may elucidate the reduced fitness of the matrix-deficient mutant strains.

Structure of concentrated nanoemulsions

We use extreme shear to create a dispersion of nanoscale droplets of silicone oil in an immiscible water phase containing an anionic surfactant sodium dodecylsulfate. Using centrifugal size fractionation, we obtain nanoemulsions having a well-defined average radius of a=75 nm. We measure the structure of concentrated nanoemulsions over a wide range of volume fractions, 0<phi<0.6, using small angle neutron scattering, and we determine the structure factor S(q) of disordered glassy dispersions of uniform deformable droplets interacting through screened surface charge repulsions. Although the low-q behavior of S(q,phi) resembles that predicted for hard spheres, the height of the primary peak does not. Instead, it exhibits a maximum as phi is increased. This difference cannot be explained solely by the droplet size polydispersity and is likely related to the deformability of the droplets that have been locked into a glassy structure.

Extreme emulsification: formation and structure of nanoemulsions

Nanoemulsions are metastable dispersions of nanodroplets of one liquid that have been ruptured by shear in another immiscible liquid. The ruptured droplets are stabilized against subsequent coalescence by a surfactant. Because the nanodroplets do not form spontaneously, as they can in lyotropic “microemulsion” phases, the structure of nanoemulsions is primarily dependent on the history of the applied shear stresses relative to the interfacial restoring stresses. By applying extremely high shear rates and controlling the composition of the emulsion, we have been able to rupture microscale droplets down to diameters as small as 30 nm in a microfluidic process that yields bulk quantities suitable for commercial production. Following ultracentrifugal fractionation to make the droplets uniform, we study the structure of these emulsions using small angle neutron scattering (SANS) at dilute and concentrated volume fractions. We contrast the structure of a concentrated nanoemulsion with the structure factor of hard spheres at a similar volume fraction.

Microfluidic synthesis of monodisperse porous microspheres with size-tunable pores

We use a perfluorinated-dendrimer–dye complex that stabilizes microbubbles as a novel pore-forming agent. We use microfluidics to produce monodisperse emulsions containing a polymer matrix material, a model active, and the perfluorinated complex; upon drying, the emulsions form porous microspheres. This porosity causes the encapsulated model active to be released faster than from non-porous microspheres. Moreover, because of the fluorous features of the pores, we can also attach an additional guest molecule to the pores which is released with a profile that is distinct from that of the encapsulated active. These porous microspheres can encapsulate and controllably release multiple actives; this makes them valuable for applications such as drug delivery and imaging.

Monodisperse Emulsion Drop Microenvironments for Bacterial Biofilm Growth

In this work, microfluidic technology is used to rapidly create hundreds of thousands of monodisperse double and triple emulsion drops that serve as 3D microenvironments for the containment and growth of bacterial biofilms. The size of these drops, with diameters from tens to hundreds of micrometers, makes them amenable to rapid manipulation and analysis. This is demonstrated by using microscopy to visualize cellular differentiation of Bacillus subtilis biofilm communities within each drop and the bacterial biofilm microstructure. Biofilm growth is explored upon specific interfaces in double and triple emulsions and upon negative and positive radii of curvature. Biofilm attachment of matrix and flagella mutants is studied as well as biofilms of Pseudomonas aeruginosa. This is the first demonstration of biofilms grown in microscale emulsion drops, which serve as both templates and containers for biofilm growth and attachment. These microenvironments have the potential to transform existing high-throughput screening methods for bacterial biofilms.

Shear-induced disruption of dense nanoemulsion gels.

The structural evolution and rheology of dense nanoemulsion gels, which have been formed by creating strong attractions between slippery nanodroplets, are explored as a function of steady shear rate using rheological small-angle neutron scattering (rheo-SANS). For applied stresses above the yield stress of the gel, the network yields, fracturing into aggregates that break and reform as they tumble and interact in the shear flow. The average aggregate size decreases with increasing shear rate; meanwhile, droplet rearrangements within the clusters, allowed by the slippery nature of the attractive interaction, increase the local density within the aggregates. At the highest shear rates, all clusters disaggregate completely into individual droplets.

Bulk manufacture of concentrated oxygen gas-filled microparticles for intravenous oxygen delivery.

Self-assembling, concentrated, lipid-based oxygen microparticles (LOMs) have been developed to administer oxygen gas when injected intravenously, preventing organ injury and death from systemic hypoxemia in animal models. Distinct from blood substitutes, LOMs are a one-way oxygen carrier designed to rescue patients who experience life-threatening hypoxemia, as caused by airway obstruction or severe lung injury. Here, we describe methods to manufacture large quantities of LOMs using an in-line, recycling, high-shear homogenizer, which can create up to 4 liters of microparticle emulsion in 10 minutes, with particles containing a median diameter of 0.93 microns and 60 volume% of gas phase. Using this process, we screen 30 combinations of commonly used excipients for their ability to form stable LOMs. LOMs composed of DSPC and cholesterol in a 1:1 molar ratio are stable for a 100 day observation period, and the number of particles exceeding 10 microns in diameter does not increase over time. When mixed with blood in vitro, LOMs fully oxygenate blood within 3.95 seconds of contact, and do not cause hemolysis or complement activation. LOMs can be manufactured in bulk by high shear homogenization, and appear to have a stability and size profile which merit further testing.

Self-assembling semiconducting polymers--rods and gels from electronic materials.

In an effort to favor the formation of straight polymer chains without crystalline grain boundaries, we have synthesized an amphiphilic conjugated polyelectrolyte, poly(fluorene-alt-thiophene) (PFT), which self-assembles in aqueous solutions to form cylindrical micelles. In contrast to many diblock copolymer assemblies, the semiconducting backbone runs parallel, not perpendicular, to the long axis of the cylindrical micelle. Solution-phase micelle formation is observed by X-ray and visible light scattering. The micelles can be cast as thin films, and the cylindrical morphology is preserved in the solid state. The effects of self-assembly are also observed through spectral shifts in optical absorption and photoluminescence. Solutions of higher-molecular-weight PFT micelles form gel networks at sufficiently high aqueous concentrations. Rheological characterization of the PFT gels reveals solid-like behavior and strain hardening below the yield point, properties similar to those found in entangled gels formed from surfactant-based micelles. Finally, electrical measurements on diode test structures indicate that, despite a complete lack of crystallinity in these self-assembled polymers, they effectively conduct electricity.

Probing phenotypic growth in expanding Bacillus subtilis biofilms

We develop an optical imaging technique for spatially and temporally tracking biofilm growth and the distribution of the main phenotypes of a Bacillus subtilis strain with a triple-fluorescent reporter for motility, matrix production, and sporulation. We develop a calibration procedure for determining the biofilm thickness from the transmission images, which is based on Beer-Lambert’s law and involves cross-sectioning of biofilms. To obtain the phenotype distribution, we assume a linear relationship between the number of cells and their fluorescence and determine the best combination of calibration coefficients that matches the total number of cells for all three phenotypes and with the total number of cells from the transmission images. Based on this analysis, we resolve the composition of the biofilm in terms of motile, matrix-producing, sporulating cells and low-fluorescent materials which includes matrix and cells that are dead or have low fluorescent gene expression. We take advantage of the circular growth to make kymograph plots of all three phenotypes and the dominant phenotype in terms of radial distance and time. To visualize the nonlocal character of biofilm growth, we also make kymographs using the local colonization time. Our technique is suitable for real-time, noninvasive, quantitative studies of the growth and phenotype distribution of biofilms which are either exposed to different conditions such as biocides, nutrient depletion, dehydration, or waste accumulation.