Microscale spatiotemporal investigation of multi-species electroactive biofilms using optical microfluidic platform – MICROBE

The scale of study envisaged is very ambitious and we will achieve this by a very original approach coupling electroanalysis methods and optomicrofluidic methods. In other words, we will design microfluidic chips on transparent electrodes and we will circulate biological liquids to form electroactive biofilms directly on the transparent electrodes.
Bacterial adhesion, colonisation and three-dimensional structuration of the biofilms on the electrodes will be monitored in real time by optical microscopy. The electrochemical activity of the adhered cells and/or biofilms will be monitored by continuous measurement of the exchange current on the electrode surface. These methods are not destructive to the biofilms, they really offer the possibility to access a dynamic monitoring of the physical structuring and the electrochemical activity of the biofilms.

The work carried out in the first part of the project (first 18 months) aimed to tackle the «scaling-down« of experiments usually involving macroscopic electrodes. This work was organised in two steps: The first was to reduce the size of the electrodes by implementing microelectrodes in large-scale electrochemical reactors. In the second stage, the complete system was reduced by developing and validating the operation of a microfluidic bioelectrochemical system.
1. Macroscopic bioelectrochemical system: reproduction of the classical behaviour of multi-species electroactive biofilms on microelectrodes
In macroscopic bioelectrochemical systems, the current density production profiles obtained for biofilms on microelectrodes were observed in the same way as for large-scale electrodes. They are characterised by an initial lag phase followed by a sharp increase to a maximum value, followed by a fall in current and a final stabilisation phase. The maximum current density was 10.3 A/m2 for salt marsh biofilms and 11.3 A/m2 for garden compost biofilms formed under polarisation for a minimum time period of 30 days. In both cases, the stainless steel microelectrodes showed exceptional repeatability and reproducibility of results compared to the Pt microelectrodes. This configuration allowed the definition of operational parameters at the microscopic scale, as well as validating the use of microelectrodes as a tool for studying phenomena that occur on larger electrodes.
2. Microscopic bioelectrochemical system: step-by-step design and experimental validation of a microfluidic and transparent bioelectrochemical cell
Laboratory-made Ag/AgCl reference electrodes were developed and tested as reference electrodes, but they did not show a stable potential for long periods of time, i.e. more than one month, in contact with biological fluids. A 2-electrode microelectrochemical system was developed using soft lithography and injection moulding techniques. A 110 µL OSTEMER polymer chip was assembled using a transparent conductive glass (ITO material) as a counter and reference pseudo-electrode, and a stainless steel microwire (Ø=50µm) as a working electrode. The design of the 2-electrode microfluidic bioelectrochemical system allows simultaneous electrical polarisation of the microelectrodes, control of the fluid inside the cell and real-time observation of the microelectrode surface with a transmission optical microscope.

Now that the small-scale BES are operational, the influence of several parameters will be investigated on the structure and performance of multi-species electroactive biofilms.
The choice of the potential applied to the electrode at the start of the formation of the electroactive biofilm will certainly have an important impact first on the physico-chemistry of the surface of the polarised material and then on the attractiveness of the material towards the bacterial cells. Subsequently, the control of the electrode potential will control the flow of electrons extracted from the biofilm.
The optimal temperature for the growth and operation of the electroactive bacterial species depends on the type of bacteria present in the inoculum. The objective will be to define the optimal temperature to ensure the growth of bacteria that could guarantee the best possible electron transfer.
The hydrodynamic conditions, especially the flow rate regime and the resulting shear stress, should help to manage the biofilm structure and the metabolic activity of the bacteria. Studies on biofilm development under various hydrodynamic conditions show that biofilm characteristics such as the amount of adhered biomass, production of extracellular polymeric substances, density and thickness vary considerably depending on the imposed shear rate.
Management of the oxygen level in the biofilm by aeration/de-aeration of the electrolyte will control the relative abundance of bacterial communities within the biofilm. In an environment without oxygen or other soluble electron acceptors, there is competition for the use of organic substrates between electroactive and fermentative populations. Microaeration of the electrolyte, during critical phases when the electroactive biofilm is less active, could limit the uncontrolled development of fermentative populations.

Stéphane Pinck, Lucila Martínez Ostormujof, Sébastien Teychené, Benjamin Erable. Microfluidic Microbial Bioelectrochemical Systems: An Integrated Investigation Platform for a More Fundamental Understanding of Electroactive Bacterial Biofilms. Microorganisms, MDPI, 2020, 8 (11), pp.1841. ?10.3390/microorganisms8111841?. ?hal-03078629?

Electroactive biofilms are electrochemical biocatalysts capable of extracting electrons (electric current) from the chemical energy contained in a wide range of organic substrates (effluents, bio-waste, biomass, etc.). They are actually tested in more than thirty applications categorized under the generic name of "bioelectrochemical systems", the best known are microbial fuel cells, biosensors or microbial electrolysis cells. The vast majority of these bioelectrochemical processes use environmental or industrial electroactive biofilms composed of a mixture of bacterial populations. This type of multi-species mixed biofilms theoretically offers the qualities of resilience and self-adaptation usually appreciated by both industrialists and academics to develop efficient and robust biological processes.
However, in the case of electroactive biofilms, the "multi-species" character confers on biofilms a complexity that nobody is able to master today. For example, electroactive biofilms contain electroactive and non-electroactive bacterial populations that interact and cooperate together. The equilibrium between these two types of populations is certainly fragile and their evolutions depend a priori on the evolution of (i) the electrode potential on which they are established, (ii) the physicochemical conditions in biofilm stratification, (ii) ) and the composition of the bulk medium (electrolyte).
It is this type of fundamental scientific questioning that we wish to answer through the MICROBE research project. Indeed, we plan to clarify at a microscopic level a whole lot of fundamental processes and mechanisms responsible for the formation and evolution of electroactive biofilms on the surface of electrodes. The scale of biofilm exploration targeted in MICROBE is very ambitious and we will access it by a very original approach coupling methods of electroanalysis and optomicrofluidic. In other words, we will design microfluidic chips on transparent electrodes and we will circulate biological fluids to form electroactive biofilms directly on the transparent electrodes.
The adhesion of the bacteria, the colonization, the three-dimensional structuring of biofilms on the electrodes will be followed in real time by optical microscopy. The electrochemical activity of the adhered cells and / or biofilms will be monitored by the continuous measurement of the exchange current on the surface of the electrodes. These methods are not destructive of biofilms, they really offer the possibility of accessing a dynamic monitoring of the physical structuring and electrochemical activity of biofilms. Other key questions such as the "dynamics of bacterial populations", the evolution of the "chemical composition" of the biofilm (essentially the exo-polymeric substances) or the "transport of soluble molecules" in the biofilm will also be informed during of the project at important moments in the life of biofilms.
Finally, we plan to explore and understand at the microscopic scale (i.e. at the scale of the biofilm) the influence of several parameters described in the literature to macroscopically impact the electrochemical activity of electroactive biofilms: quorum sensing, electrons flow, electrolyte composition and hydrodynamics.
To carry out the proposed systemic approach described in MICROBE, we have gathered complementary skills in microfluidics, electroanalysis, microscopy, microsystems and ecology of microbial communities around a consortium of partners already experienced on the topic of electroactive biofilms (LGC Toulouse, LETI Grenoble and LEMIRE Cadarache).