• Energy Research

Abstract Microbial fuel cells (MFCs) have applications other than electricity production, including the capacity to power desirable reactions in the cathode chamber. However, current knowledge of the microbial ecology and physiology of biocathodes is minimal, and as a result more research dedicated to understanding the microbial communities active in cathode biofilms is required. Here we characterize the microbiology of denitrifying bacterial communities stimulated by reducing equivalents generated from the anodic oxidation of acetate. We analyzed biofilms isolated from two types of cathodic denitrification systems: (1) a loop format where the effluent from the carbon oxidation step in the anode is subjected to a nitrifying reactor which is fed to the cathode chamber and (2) an alternative non-loop format where anodic and cathodic feed streams are separated. The results of our study indicate the superior performance of the loop reactor in terms of enhanced current production and nitrate removal rates. We hypothesized that phylogenetic or structural features of the microbial communities could explain the increased performance of the loop reactor. We used PhyloChip with 16S rRNA (cDNA) and fluorescent in situ hybridization to characterize the active bacterial communities. Our study results reveal a greater richness, as well as an increased phylogenetic diversity, active in denitrifying biofilms than was previously identified in cathodic systems. Specifically, we identified Proteobacteria, Firmicutes and Chloroflexi members that were dominant in denitrifying cathodes. In addition, our study results indicate that it is the structural component, in terms of bacterial richness and evenness, rather than the phylogenetic affiliation of dominant bacteria, that best corresponds to cathode performance.

Abstract Reduction of CO2 to acetate in microbial electrosynthesis has been widely studied. However, the selective and quantitative production of longer chain chemicals and biofuels is still a bottleneck. Lack of sufficient energy provided by only the cathode electrode in Bio-electrochemical systems during chain elongation is one of the key challenges. It is assumed that additional electron donors than a polarised cathode is required to steer the production towards longer chain of carboxylates than acetate. In this study, formate and ethanol were supplied separately in the reactors fed by CO2 for 45 days in addition to the cathodes poised at −1.0 V vs. Ag/AgCl to investigate their effect on production. Although acetate was still the major product, supplying electron donors directed the production towards more diverse and longer chain organic chemicals than that in presence of the polarised cathode only. Significant improvement in the production of butyrate (×3.8 increase in maximum concentration) and butanol (maximum of 6.8 ± 0.3 mmol C L−1) was observed after supplying formate, while ethanol increased the diversity of the products. Supplying formate and ethanol in reactors for another 30 days under open circuit potential clarified that only ethanol could provide sufficient energy for butyrate production from acetate in the absence of polarised cathode, which reached the highest butyrate concentration of 19.1 ± 2.3 mmol C L−1. Formate was only consumed in presence of polarised cathode. It is proposed in our study that production of C4 products in presence of only cathodic electrode or cathodic electrode and formate could be associated to initial reduction of acetate to ethanol, consumed for production of C4 products through acetate. Trace levels of caproate and hexanol were detected in both reactors supplied with formate and ethanol only in the presence of polarised cathode.

Microbial electrosynthesis is a novel approach that aims at shifting the cellular metabolism towards electron-dense target products by extracellular electron supply. Many organisms including several acetogenic bacteria have been shown to be able to consume electrical current. However, suitable hosts for relevant industrial processes are yet to be discovered, and major knowledge gaps about the underlying fundamental processes still remain.In this paper, we present the first report of electron uptake by the Gram-positive, ethanol-producing acetogen, Clostridium autoethanogenum. Under heterotrophic conditions, extracellular electron supply induced a significant metabolic shift away from acetate. In electrically enhanced fermentations on fructose, acetate production was cut by more than half, while production of lactate and 2,3-butanediol increased by 35-fold and threefold, respectively. The use of mediators with different redox potential revealed a direct dependency of the metabolic effect on the redox potential at which electrons are supplied. Only electrons delivered at a redox potential low enough to reduce ferredoxin caused the reported effect.Production in acetogenic organisms is usually challenged by cellular energy limitations if the target product does not lead to a net energy gain as in the case of acetate. The presented results demonstrate a significant shift of carbon fluxes away from acetate towards the products, lactate and 2,3-butanediol, induced by small electricity input (~0.09 mol of electrons per mol of substrate). This presents a simple and attractive method to optimize acetogenic fermentations for production of chemicals and fuels using electrochemical techniques. The relationship between metabolic shift and redox potential of electron feed gives an indication of possible electron-transfer mechanisms and helps to prioritize further research efforts.

Electrochemically active biofilms rely on microorganisms capable of extracellular electron transfer. Such biofilms are involved in the dissimilatory reduction of metal oxides in natural environments as well as electricity driving and driven processes at the electrodes of microbial bioelectrochemical systems. In this work we present the application of confocal Raman microscopy (CRM) as a non-invasive, label-free, and in vivo characterization method of acetate oxidizing anodic biofilms, grown from primary wastewater inoculum and dominated by Geobacter species (>85% of sequences analysed using pyrotag sequencing). Using the resonance Raman effect of the heme protein cytochrome c (Cyt c)—an ubiquitous component of extracellular electron transfer reactions—it was possible to collect characteristic spectral information of electrochemically active biofilms at pixel integration times of 0.2 s and an excitation wavelength of 532 nm. This allowed monitoring of biofilm development at different growth stages, without impacting its structural or metabolic activity. Furthermore, we demonstrate the possibility of non-invasive investigation of the spatial redox electrochemistry (up to a compositional level) of electrochemically active biofilms, as we observed significant changes in the vibrational properties of Cyt c resulting from shifts in the anodic potential between different redox conditions. Compared to conventional methods requiring destructive sample manipulation and fixation, the proposed approach based on CRM allows the non-invasive analysis of microbial aggregates with minimal sample preparation or prior knowledge of the sample.

Quantitative proteomics from low biomass, biofilm samples is not well documented. In this study we show successful use of SWATH-MS for quantitative proteomic analysis of a microbial electrochemically active biofilm. Shewanella oneidensis MR-1 was grown on carbon cloth electrodes under continuous anodic electrochemical polarizations in a bioelectrochemical system (BES). Using lactate as the electron donor, anodes serving as terminal microbial electron acceptors were operated at three different electrode potentials (+0.71 V, +0.21 V & -0.19 V vs. SHE) and the development of catalytic activity was monitored by measuring the current traces over time. Once maximum current was reached (usually within 21-29 h) the electrochemical systems were shut off and biofilm proteins were extracted from the electrodes for proteomic assessment. SWATH-MS analysis identified 704 proteins, and quantitative comparison was made of those associated with tricarboxcylic acid (TCA) cycle. Metabolic differences detected between the biofilms suggested a branching of the S. oneidensis TCA cycle when grown at the different electrode potentials. In addition, the higher abundance of enzymes involved in the TCA cycle at higher potential indicates an increase in metabolic activity, which is expected given the assumed higher energy gains. This study demonstrates high numbers of identifications on BES biofilm samples can be achieved in comparison to what is currently reported. This is most likely due to the minimal preparation steps required for SWATH-MS.

Microbial fuel cells (MFCs) can use nitrate as a cathodic electron acceptor, allowing for simultaneous removal of carbon (at the anode) and nitrogen (at the cathode). In this study, we supplemented the cathodic process with in situ nitrification through specific aeration, and thus obtained simultaneous nitrification and denitrification (SND) in the one half-cell. Synthetic wastewater containing acetate and ammonium was supplied to the anode; the effluent was subsequently directed to the cathode. The influence of oxygen levels and carbon/nitrogen concentrations and ratios on the system performances was investigated. Denitrification occurred simultaneously with nitrification at the cathode, producing an effluent with levels of nitrate and ammonium as low as 1.0+/-0.5 mg N L(-1) and 2.13+/-0.05 mg N L(-1), respectively, resulting in a nitrogen removal efficiency of 94.1+/-0.9%. The integration of the nitrification process into the cathode solves the drawback of ammonium losses due to diffusion between compartments in the MFC, as previously reported in a system operating with external nitrification stage. This work represents the first successful attempt to combine SND and organics oxidation while producing electricity in an MFC.

A microbial electrosynthesis cell comprising two biological cathode chambers sharing the same anode compartment is used to promote the production of C2–C4 carboxylic acids and alcohols from carbon dioxide.

AbstractMethanol is a promising renewable energy carrier that can be used as a favourable substrate for biotechnology, due to its high energy efficiency conversion and ease of integration within existing infrastructure. Some acetogenic bacteria have the native ability to utilise methanol, along with other C1substrates such as CO2and formate, to produce valuable chemicals. Continuous cultures favour economically viable bioprocesses, however, the performance of acetogens has not been investigated at the molecular level when grown on methanol. Here we present steady-state chemostat quantification of the metabolism ofEubacterium limosum, finding maximum methanol uptake rates up to 640±22 mmol/gDCW/d, with significant fluxes to butyrate. To better understand metabolism of acetogens under methanol growth conditions, we sampled chemostats for proteomics and metabolomics. Changes in protein expression and intracellular metabolomics highlighted key aspects of methanol metabolism, and highlighted bottleneck conditions preventing formation of the more valuable product, butanol. Interestingly, a small amount of formate in methylotrophic metabolism triggered a cellular state known in other acetogens to correlate with solventogenesis. Unfortunately, this was prevented by post-translation effects including an oxidised NAD pool. There remains uncertainty around ferredoxin balance at the methylene-tetrahydrofolate reductase (MTHFR) and at the Rnf level.