Development of a robust microfluidic electrochimical cell for biofilm study in controlled hydrodynamic conditions

Le domaine de la bioélectrochimie a actuellement un grand impact sur les nouvelles biotechnologies, notamment les dispositifs médicaux aux points de service et la détection bioélectrochimique. D'autre part, les systèmes émergents de bioénergie offrent de nouvelles opportunités pour se passer des produits pétroliers classiques grâce à des approches alternatives plus durables sur le plan environnemental. En tant que telle, la branche de la bioélectrochimie traitant des systèmes énergétiques est sur le point d’avoir un impact incontestable sur les concepts d’énergie verte et de bioénergie. Pour faciliter ces études et d'autres, les systèmes bioélectrochimiques (BES), qui utilisent des composants biologiques tels que des bactéries (souvent appelées biocatalyseurs), sont de plus en plus développés et miniaturisés pour une nouvelle série de biotechnologies. Cette thèse porte sur la fabrication et la fonctionnalité d’un « système microfluidique électrochimique à trois électrodes » pour l’étude de biofilms de différentes bactéries (électroactives et non-électroactives) à l’aide de différentes techniques électrochimiques. Ces biofilms ont été largement étudiés par des techniques électrochimiques et d’imagerie microscopique (microscopie optique et électronique). Cette thèse pourra potentiellement ouvrir la voie à une nouvelle vague de développements de biocapteurs électrochimiques, tout en offrant des avancées scientifiques spécifiques dans les études de biocapacité de biofilm, de biorésistance, de pH du biofilm, de dépendance nutritionnelle de l'activité du biofilm et de la cinétique de respiration bactérienne.The area of bioelectrochemistry is currently making the greatest impact in new biotechnology, including point of care medical devices and bioelectrochemical sensing. On the other hand, emerging bioenergy systems offer new opportunities to move away from conventional petroleum products toward more environmentally sustainable alternative approaches. As such, the branch of bioelectrochemistry dealing with energy systems is poised to have an undoubtable impact on greenenergy and bioenergy concepts. To facilitate these and other areas of study, bioelectrochemical systems (BESs), which use biological components such as bacteria (often referred to as biocatalysts) are increasingly being developed and miniaturized for a new round of biotechnology. This PhD thesis focuses on fabrication and functionality of a “three-electrode electrochemical microfluidic system” for biofilm studies of different bacteria (electroactive and non-electroactive) using different electrochemical techniques. They were broadly studied by electrochemical and microscopic imaging (optical and electron microscopy) techniques. This thesis can potentially open the way for a new wave of electrochemical biosensor development, while offering specific scientific advances in studies of biofilm biocapacitance, bioresistance, biofilm pH, nutrient dependency of biofilm activity and bacterial respiration kinetics

A generalized kinetic framework applied to whole-cell bioelectrocatalysis in bioflow reactors clarifies performance enhancements for geobacter sulfurreducens biofilms

A common kinetic framework for studies of whole‐cell catalysis is vital for understanding and optimizing bioflow reactors. In this work, we demonstrate the applicability of a flow‐adapted version of Michaelis‐Menten kinetics to an electrocatalytic bacterial biofilm. A three‐electrode microfluidic biofilm flow reactor measured increased turnover rates by as much as 50 % from a Geobacter sulfurreducens biofilm as flow rate was varied. Based on parameters from the applied kinetic framework, flow‐induced increases to turnover rate, catalytic efficiency and device reaction capacity could be linked to an increase in catalytic biomass. This study demonstrates that a standardized kinetic framework is critical for quantitative measurements of new living catalytic systems in flow reactors and for benchmarking against well‐studied catalytic systems such as enzymes

Microfluidic flow confinement to avoid chemotaxis-based upstream growth in a biofilm flow cell reactor

Introduction of a bacterial inoculant into a chemostat bioreactor can lead to unwanted contamination of upstream elements via chemotaxis. This can result in biofilm growth in connective tubing, valves and even the medium source reservoir itself, thus complicating the conditions of the applied liquid phase and impeding proper chemostat functionality. Applied to biofilm forming Pseudomonas fluorescens bacteria, we tested two different microfluidic flow confinement methods designed to impede upstream contamination. The first isolated biofilm growth from the relatively stagnant zones within the microchannel corners, and in the second a flow enhancement element was introduced to increase flow velocities and shear forces. Both methods showed improvement over a control design, but flow enhancement showed the best performance by delaying or preventing bacterial contamination of upstream elements, ensuring stability of the applied liquid media conditions for the entire duration of the experiments. This simple passive element has the potential for wide use as it is easy to implement and can be optimised for different experimental requirements

Microfluidic bioanalytical flow cells for biofilm studies : a review

Bacterial biofilms are among the oldest and most prevalent multicellular life forms on Earth and are increasingly relevant in research areas related to industrial fouling, medicine and biotechnology. The main hurdles to obtaining definitive experimental results include time-varying biofilm properties, structural and chemical heterogeneity, and especially their strong sensitivity to environmental cues. Therefore, in addition to judicious choice of measurement tools, a well-designed biofilm study requires strict control over experimental conditions, more so than most chemical studies. Due to excellent control over a host of physiochemical parameters, microfluidic flow cells have become indispensable in microbiological studies. Not surprisingly, the number of lab-on-chip studies focusing on biofilms and other microbiological systems with expanded analytical capabilities has expanded rapidly in the past decade. In this paper, we comprehensively review the current state of microfluidic bioanalytical research applied to bacterial biofilms and offer a perspective on new approaches that are expected to drive continued advances in this field

A microfluidic method and custom model for continuous, non-intrusive biofilm viscosity measurements under different nutrient conditions

Straight, low-aspect ratio micro flow cells are used to support biofilm attachment and preferential accumulation at the short side-wall, which progressively reduces the effective channel width. The biofilm shifts downstream at measurable velocities under the imposed force from the constant laminar co-flowing nutrient stream. The dynamic behaviour of the biofilm viscosity is modeled semi-analytically, based on experimental measurements of biofilm dimensions and velocity as inputs. The technique advances the study of biofilm mechanical properties by strongly limiting biases related to non-Newtonian biofilm properties (e.g., shear dependent viscosity) with excellent time resolution. To demonstrate the proof of principle, young Pseudomonas sp. biofilms were analyzed under different nutrient concentrations and constant micro-flow conditions. The striking results show that large initial differences in biofilm viscosities grown under different nutrient concentrations become nearly identical in less than one day, followed by a continuous thickening process. The technique verifies that in 50 h from inoculation to early maturation stages, biofilm viscosity could grow by over 2 orders of magnitude. The approach opens the way for detailed studies of mechanical properties under a wide variety of physiochemical conditions, such as ionic strength, temperature, and shear stress

A high performance membraneless microfluidic microbial fuel cell for stable, long-term benchtop operation under strong flow

Strong control over experimental conditions in microfluidic channels provides a unique opportunity to study and optimize membraneless microbial fuel cells (MFCs), particularly with respect to the role of flow. However, improved performance and transferability of results to the wider MFC community require improvements to device stability under all applied operational conditions. To address these challenges, we present an easy-to-fabricate membraneless MFC that combines i) O2 protection via a gas diffusion barrier, ii) integrated graphite electrodes, and iii) optimized electrode placement to avoid cross-contamination under all applied flow rates. Attention to all of these design features in the same platform resulted in the operation of a MFC with a pure-culture anaerobic Geobacter sulfurreducens biofilm for half a year, that is, six times longer than previously reported, without the use of an oxygen scavenger. As a result of higher device stability under high flow rates, power densities were four times higher than reported previously for microfluidic MFCs with the same biofilm

Microstructured anodes by surface wrinkling for studies of direct electron transfer biofilms in microbial fuel cells

A method for producing hierarchical wrinkled gold surfaces is used to continuously change characteristic microstructure dimensions of a bioanode in a microbial fuel cell, while conserving the total electroactive surface area and material chemistry. Using this approach, the effect of anode topography on power outputs from direct electron transfer from Geobacter sulfurreducens biofilms can be isolated and studied without the competing effects associated with additive manufacturing. Despite having the same electroactive surface area for all structured anodes, tall and well‐spaced features perform best. Anodes with the shortest, most closely packed structures, on the other hand, do not perform any better than planar surfaces with the same footprint and lower electroactive surface area. It is postulated that large interfold spacing provides better electrical contact between the biofilm and the electrode via improved bacterial packing density at the electrode surface. Rigorous attention to structural dimensions rather than total electroactive surface area is proposed as an important direction for future bioanode optimization in microbial fuel cells containing direct electron transfer electroactive biofilms