Microbial electrochemical technologies (METs) are promising for sustainable applications. Recently, electron storage during intermittent operation of electroactive biofilms (EABs) has been shown to play an important role in power output and electron efficiencies. Insights into electron storage mechanisms, and the conditions under which these occur, are essential to improve microbial electrochemical conversions and to optimize biotechnological processes. Here, we discuss the two main mechanisms for electron storage in EABs: storage in the form of reduced redox active components in the electron transport chain and in the form of polymers. We review electron storage in EABs and in other microorganisms and will discuss how the mechanisms of electron storage can be influenced. Controlling Electron Flows in EABs for High Electron EfficiencyTo safeguard the Earth's resources for future generations, it is of utmost importance to reduce our CO 2 footprint and to recover valuable components from waste streams for reuse. To address this huge challenge, it is essential to develop and mature sustainable technologies. Microbial electrochemical technologies (METs) have promising applications in resource and energy recovery and bioremediation [1][2][3][4]. METs collectively refer to systems that use a combination of electrodes and electroactive biofilms (EABs) (see Glossary) for different biological conversions. The key property of these EABs is that they can transfer electrons between chemical bonds and electrodes, which can serve many different applications [5,6] (Box 1).

Detailed studies of microbial growth in bioelectrochemical systems (BESs) are required for their suitable design and operation. Here, we report the use of optical coherence tomography (OCT) as a tool for in situ and noninvasive quantification of biofilm growth on electrodes (bioanodes). An experimental platform is designed and described in which transparent electrodes are used to allow real‐time, 3D biofilm imaging. The accuracy and precision of the developed method is assessed by relating the OCT results to well‐established standards for biofilm quantification (chemical oxygen demand (COD) and total N content) and show high correspondence to these standards. Biofilm thickness observed by OCT ranged between 3 and 90 μm for experimental durations ranging from 1 to 24 days. This translated to growth yields between 38 and 42 mgCODbiomass  gCODacetate −1 at an anode potential of −0.35 V versus Ag/AgCl. Time‐lapse observations of an experimental run performed in duplicate show high reproducibility in obtained microbial growth yield by the developed method. As such, we identify OCT as a powerful tool for conducting in‐depth characterizations of microbial growth dynamics in BESs. Additionally, the presented platform allows concomitant application of this method with various optical and electrochemical techniques.

Electro-active bacteria (EAB) can form biofilms on an anode (so-called bioanodes), and use the electrode as electron acceptor for oxidation of organics in wastewater. So far, bioanodes have mainly been investigated under a continuous anode potential, but intermittent anode potential has resulted in higher currents and different biofilm morphologies. However, little is known about how intermittent potential influences the electron balance in the anode compartment. In this study, we investigated electron balances of bioanodes at intermittent anode potential regimes. We used a transparent non-capacitive electrode that also allowed for in-situ quantification of the EAB using optical coherence tomography (OCT). We observed comparable current densities between continuous and intermittent bioanodes, and stored charge was similar for all the applied intermittent times (5 mC). Electron balances were further investigated by quantifying Extracellular Polymeric Substances (EPS), by analyzing the elemental composition of biomass, and by quantifying biofilm and planktonic cells. For all tested conditions, a charge balance of the anode compartment showed that more electrons were diverted to planktonic cells than biofilm. Besides, 27–43% of the total charge was detected as soluble EPS in intermittent bioanodes, whereas only 15% was found as soluble EPS in continuous bioanodes. The amount of proteins in the EPS of biofilms was higher for intermittent operated bioanodes (0.21 mg COD proteins mg COD biofilm −1 ) than for continuous operated bioanodes (0.05 mg COD proteins mg COD biofilm −1 ). OCT revealed patchy morphologies for biofilms under intermittent anode potential. Overall, this study helped understanding that the use of a non-capacitive electrode and intermittent anode potential deviated electrons to other processes other than electric current at the electrode by identifying electron sinks in the anolyte and quantifying the accumulation of electrons in the form of EPS.

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