The diversity of techniques to study electrochemically active biofilms highlights the need for standardization

Microbial bioelectrochemical systems (BESs) employ whole microorganisms to catalyze electrode reactions. BESs allow electricity generation from wastewater, electricity-driven (bio)production, biosensing, and bioremediation. Many of these processes are perceived as highly promising; however, to date the performance of particularly bioproduction processes is not yet at the level required for practical applications. Critical to enabling high catalytic activity are the electrochemically active microorganisms. Whether the biocatalyst comes as a planktonic cell, a surface monolayer of cells, or a fully developed biofilm, effective electron transfer and process performance need to be achieved. However, despite many different approaches and extensive research, many questions regarding the functioning of electroactive microorganisms remain open. This is certainly due to the complexity of bioelectrochemical processes, as they depend on microbial, electrochemical, physical-chemical, and operational considerations. This versatility and complexity calls for a plethora of analytical tools required to study electrochemically active microorganisms, especially biofilms. Here, we present an overview of the parameters defining electroactive microbial biofilms (EABfs) and the analytical toolbox available to study them at different levels of resolution. As we will show, a broad diversity of techniques have been applied to this field; however, these have often led to conflicting information. Consequently, to alleviate this and further mature the field of BES research, a standardized framework appears essential.