Defossilization of industrial processes has led to a growing interest in alternative biotechnologies capable of producing chemicals from renewable resources. Microbial electrosynthesis (MES) is an emerging technology in which electrotrophic microorganisms utilize electrons from a cathode and CO₂ to produce multi-carbon compounds. To reach industrial application, clearer insights into the interactions between underlying biological, electrochemical, and physicochemical processes are required. Although individual parameters have been widely studied, identifying the most influential factors and their interactions remains challenging. This study applies design of experiments (DoE) and mixed linear regression modeling (MLRM) to examine the influence of pH, CO₂ and H₂ partial pressures, acetic acid concentration, and the addition of tungsten and selenium on the production spectrum in biofilm-driven MES. The developed DoE-MLRM approach highlights the key role of pH and CO₂ availability in supporting carbon fixation and acetate production, while the trace metals selenium and tungsten mostly promote chain elongation.

Carbon capture and utilisation are crucial for reducing fossil fuel dependence and transforming the chemical and energy industries. Microbial electrosynthesis (MES) is a promising technology where electrotrophic microorganisms convert CO₂ into valuable biochemicals using electricity. Despite recent advancements, replicability in MES remains poorly understood, with scarce pre-inoculation abiotic data and limited exploration of abiotic and biotic performance correlations. This study introduces a novel miniaturised reactor, modelled after a state-of-the-art flat-plate directed-flow-through bioelectrochemical reactor (DFBR). Four miniaturised reactors were tested in parallel under abiotic conditions to evaluate the impact of electrode material, reactor design, and assembly on replicability of electrochemical behaviour. Using the dynamic time warping (DTW) algorithm, reactor similarity was quantified for the first time based on electrochemical performance. Kernel scatterplot smoothing on micro-CT data revealed that electrodes, particularly the commonly used carbon felt, are a significant source of variability in electrochemical performance, as further supported by additional abiotic electrochemical tests. Additionally, the miniaturised reactors were inoculated with an enriched mixed culture to examine microbial activity's effect on replicability, achieving concentrations up to 4.55 g L^-1 acetate, 0.96 g L^-1 butyrate, and 0.38 g L^-1 caproate after 60 days. Variations in abiotic conditions, including maximum reachable current density, onset potential, and porosity, influence biofilm growth and performance. The miniaturised DFBR effectively represents the serpentine DFBR, while the adaptable reactor design and proposed statistical methods set a new benchmark for MES research.

The practical implementation of microbial electrosynthesis (MES) is currently limited by the slow microbial colonisation of the electrode and the need to suppress methanogenic activity. This study investigates a two-stage strategy to suppress methanogenesis and promote the rapid formation of an acetogenic biofilm in a directed-flow-through bioelectrochemical reactor. Four start-up regimes were compared: mixotrophic without heat pre-treatment (M), mixotrophic with heat pre-treatment (MT), heterotrophic without heat pre-treatment (H), and heterotrophic with heat pre-treatment (HT), each followed by a common autotrophic phase. Mixotrophy outperformed heterotrophy by accelerating and increasing acetate accumulation. However, adding heat pre-treatment (MT) introduced a short lag phase and resulted in less sustained chain elongation than mixotrophy alone (M). Under the mixotrophic regime, microbial analysis showed an enrichment of genera with acetogenic representatives such as Clostridium sensu stricto 12 and Sporomusa, alongside a reduction in facultative anaerobic and fermentative bacteria. Full biofilm colonisation of the electrode was achieved within 55 to 65 days, while acetate, butyrate, and caproate production was initiated within the first week, reaching concentrations typically observed only after approximately 70 days under autotrophic conditions. Methane remained undetectable for about 40 days and, when detected later, exhibited low coulombic efficiencies (< 1%). Taken together, these results indicate that mixotrophic start-up provides a promising route to accelerate electrode colonisation and enhance early-stage productivity in MES, while highlighting the need for further optimisation and a deeper understanding of microbial interactions.