www.nature.com/npjbiofilms ARTICLE OPEN Parameters influencing the development of highly conductive and efficient biofilm during microbial electrosynthesis: the importance of applied potential and inorganic carbon source ✉ Paniz Izadi1, Jean-Marie Fontmorin1, Alexiane Godain 2, Eileen H. Yu1,3 and Ian M. Head2 Cathode-driven applications of bio-electrochemical systems (BESs) have the potential to transform CO2 into value-added chemicals using microorganisms. However, their commercialisation is limited as biocathodes in BESs are characterised by slow start-up and low efficiency. Understanding biosynthesis pathways, electron transfer mechanisms and the effect of operational variables on microbial electrosynthesis (MES) is of fundamental importance to advance these applications of a system that has the capacity to convert CO2 to organics and is potentially sustainable. In this work, we demonstrate that cathodic potential and inorganic carbon source are keys for the development of a dense and conductive biofilm that ensures high efficiency in the overall system. Applying the cathodic potential of −1.0 V vs. Ag/AgCl and providing only gaseous CO2 in our system, a dense biofilm dominated by Acetobacterium (ca. 50% of biofilm) was formed. The superior biofilm density was significantly correlated with a higher production yield of organic chemicals, particularly acetate. Together, a significant decrease in the H2 evolution overpotential (by 200 mV) and abundant nifH genes within the biofilm were observed. This can only be mechanistically explained if intracellular hydrogen production with direct electron uptake from the cathode via nitrogenase within bacterial cells is occurring in addition to the 1234567890():,; commonly observed extracellular H2 production. Indeed, the enzymatic activity within the biofilm accelerated the electron transfer. This was evidenced by an increase in the coulombic efficiency (ca. 69%) and a 10-fold decrease in the charge transfer resistance. This is the first report of such a significant decrease in the charge resistance via the development of a highly conductive biofilm during MES. The results highlight the fundamental importance of maintaining a highly active autotrophic Acetobacterium population through feeding CO2 in gaseous form, which its dominance in the biocathode leads to a higher efficiency of the system. npj Biofilms and Microbiomes (2020) 6:40 ; https://doi.org/10.1038/s41522-020-00151-x INTRODUCTION product obtained in MES systems13. In addition, chain elongation Over the last few decades, fossil fuels have been used as the main from acetate for production of longer chain organics such as source of energy for human and industrial activities. The intense butyrate, caproate and their corresponding alcohols by MES from 14–22 consumption of these resources has led to significant environ- CO2 has been reported recently . Despite the growing number 1 mental effects including global warming and climate change .CO2 of studies on MES, major bottlenecks exist in the development emissions to the atmosphere from fossil fuel combustion is and scale up of the technology. One important limitation is the 2 believed to be the main driver of these effects . A range of slow development of CO2-reducing biofilms on cathodes related approaches, including electrochemical methods, have been to the low growth rate and yield of bacteria growing on the 3 developed to reduce CO2 to valuable chemicals . Over the past electrode surface and using the electrode as an electron donor few decades, metal catalysts such as copper have been commonly and CO2 as a carbon source. This leads to the slow or sometimes fi used for electrochemical reduction of CO2 and have received lots no bio lm formation and thus slow start-up typically observed in of attention in this area4–7. Microbial electrosynthesis (MES) is a MES processes23,24. In addition, the nature of the mechanisms (e.g. bio-electrochemical technique that has offered the sustainable direct and indirect) involved in extracellular electron transfer from conversion of CO2 to valuable organic chemicals at the cathode of the electrode to microorganisms and the role of planktonic cells in bio-electrochemical systems (BES), using microorganisms as MES have not been fully established. Several assumptions were biocatalysts8–10. Despite the fact that electrochemical processes made in previous studies, suggesting that MES processes would for CO2 reduction to date are much more mature and closer to either be driven by planktonic bacterial community in the industrial applications compared to MES, MES offers other catholyte25, or only by the cathodic biofilm which would be 19 advantages that are worth being considered such as lower over responsible for CO2 conversion . Through the direct interaction of potential, and therefore lower energy consumption and more microbial cells with the cathode, it was proposed that electrons sustainable catalysts. In addition, the products in electrochemical are transferred directly from the cathode to the microbial fi 8,26–28 fi CO2 reduction are typically limited to lower carbon products, community in the cathodic bio lm . However, no speci c particularly C1 compounds, formate and CO11,12. However, MES pathway of direct electron transfer (DET) has been identified in offers the opportunity to convert CO2 to more complex MES systems. Nevertheless, indirect electron transfer (IET) has carbonaceous organic products. Currently, acetate synthesis been observed in many studies, mainly with H2 as an intermediate through the Wood–Ljungdahl pathway has been the major electron carrier28,29. In previous studies on pure and mixed 1School of Engineering, Newcastle University, Newcastle upon Tyne, UK. 2School of Natural and Environmental Sciences, Newcastle upon Tyne, UK. 3Department of Chemical ✉ Engineering, Loughborough University, Loughborough, UK. email: [email protected] Published in partnership with Nanyang Technological University P. Izadi et al. 2 cultures of microorganisms, a potential of −0.6 V vs. Ag/AgCl was morphological properties of the mature biofilm formed during MES applied at the cathode to discard the possibility of abiotic H2 processes as well as the microbial composition of the biofilm were production. It was suggested that in the absence of H2 as an studied. The potential mechanisms of electron transfer between the intermediate electron carrier, acetogens such as Sporomusa ovata cathode and microbial cells and organic compound synthesis were and Clostridium ljungdahlii were able to take up electrons directly also investigated by shotgun metagenome sequencing. from the cathode30–32. However, even at −0.6 V it appears that electrons are still transferred between the cathode and CO - 2 RESULTS AND DISCUSSION reducing microorganisms such as methanogens through H2, produced enzymatically at the surface of the cathode, and Effect of cathode potential on organic compounds production by consumed quickly by cathodic microorganisms33. The cathode MES potential is, therefore, a key parameter affecting the mechanism of Current consumption and acetate production from CO2 at cathodic 34 electron transfer in MES systems .AsH2 production increases the potentials of −1.0 V and −0.8 V (BES 1 and BES 3). Before starting electron transfer rate, rather than DET, production of medium and BES, an abiotic control experiment was carried out for 13 days to long chain organic compounds has been achieved in a number of evaluate the possible production of H2 at −1.0 V (BES 1) and studies by applying a negative potential (commonly more −0.8 V (BES 3). CO2 was the carbon source in both conditions. The negative than −0.7 V vs. Ag/AgCl) at the cathode19–21,35. However, cathodic current during abiotic control was around −0.01 and only a small number of these studies reported the formation of a −0.03 mA cm−2 at the applied potential of −0.8 and 1.0 V, 19 cathodic biofilm in such conditions . Dense and well-developed respectively (Supplementary Fig. 1). Subsequently, no H2 was cathodic biofilms have been shown to significantly improve detected in the headspace of the reactors poised at −0.8 V over a 36 electron transfer rates and production rates in MES , while lower 13 day period, however, 1.3 ± 0.3 ml H2 was detected in the production rates were observed in MES where planktonic cells headspace of reactors at −1.0 V. No other gas products were dominated product formation37. In addition, MES driven by detected in the headspaces of the reactors. The possibility of planktonic cells has the limitation that microbial communities abiotic H2 evolution on a graphite felt cathode poised at −1.0 V can be washed out from the reactor by changing the operational was confirmed by CV of a plain graphite felt electrode (see CV mode to continuous feeding regime25. A significant increase (from results). In addition, no organic products were detected in the 2.3 up to 11.8 fold) in acetate production rate was reported after catholyte, showing that abiotic reduction of CO2 does not occur at the development of a dense S. ovata biofilm using a modified these potentials. After starting the biotic experiment, to test the 36,38–41 1234567890():,; electrode . However, number of studies have shown that effect of cathode potential on current consumption and acetate only thin, monolayer biofilms can form on un-modified electro- production through MES, two reactors, BES 1 and BES 3, were des42,43. Due to the difficulties of enrichment of desirable bacteria operated at −1.0 V and −0.8 V, respectively for 104 days. Effluent from diverse microbial communities, it is essential to study the from an operating BES biocathode, producing acetate as the sole formation of well-developed biofilms from mixed inocula for MES product (7.5 ± 1.1 mM), was used as inoculum. In each freshly to improve the applicability of this technique. In addition, in inoculated BES, cathodic current started within 1 day of anodic biofilms typically dominated by Geobacter, cytochromes inoculation, most significantly in BES 1 (−1.0 V/CO2). A maximum have been shown to play an important role in enhancing current density obtained in BES with different cathode potentials conductivity of the biofilm and subsequently the efficiency of ranged from –0.007 to –1.8 mA cm−2 (Supplementary Fig.