Exoelectrogenic biofilm growth in shearing flows

Abstract

Microbial biofouling occurs when a biofilm adheres to materials involved in liquid transport causing economic loss through corrosion and drag losses on ship hulls, and in oil and food distribution. Microorganisms interacting with surfaces under these open channel flows contend with high shear rates and active transport to the surface. The metallic surfaces they interact with carry charge at various potentials that are little addressed in literature. We demonstrate for the first time that mass transport limiting current, chronoamperometry, and cyclic voltammetry in a rotating disk electrode are ideal for studying adhesion of microbes to metallic surfaces under shear. We study the adhesion of Escherichia coli, Bacillus subtilis, and 1 μm silica microspheres over a range of shear stresses. Our results agree with literature on red blood cells in rotating disk electrodes and deposition rates of B. subtilis and E. coli from optical systems, and show that we can quantify changes in active electrode area by bacteria adhesion and protein secretion.

Our methodology measures changes in area instead of mass simultaneously providing measurements of the protein binding step that initiates biofilm formation. Unlike fluorescence microscopy, these methods are in vivo and apply to a larger range of problems than on-chip flow devices. We also use the rotating disk system to present the first study of how electroactive biofilms adapt to shear stress over time. These biofilms are unique in that they do not rely on electron acceptor diffusion as they are "wired" to the electron acceptor, leading to thicker biofilms. Furthermore, it is possible to use the current produced by the biofilms as a proxy for metabolic respiration. We measure current, open circuit potential, electron diffusion current, electrochemical impedance, and formal potential throughout the course of seven days of Geobacter sulfurreducens forming a biofilm on a graphite disk exposed to three different shear stresses (1, 0.1, 0.01 Pa) and fixed mass flux. We image the resulting biofilm to measure biofilm thickness, porosity, and surface roughness. We find that high shear rates lead to faster start-up times and higher current, and by proxy higher metabolic rates, at the cost of long term sustainability of this current. We also find that there was no statistical difference in thickness or surface roughness between biofilms of different stresses. Similar to previous work, we propose that the lack of stability is due to the absence of waste removal. Our results are the first to show that the rotating disk system can be used investigate biofilm's development, metabolism, and structure from initiation to decay in vivo under fluid shear stress and electrical stress conditions that occur in our engineered environments. Future work using this system can include increased sampling frequency to understand start-up behavior and analysis of how mixed cultures modify adhesion, start-up respiration rates, and waste removal.