Search

Large-scale environmental applications of microbial electrochemical technologies (MET), such as wastewater treatment, bioremediation, or soil improvement, would be more feasible if bioelectrodes could be fabricated with simpler materials. Biochar with potentially improved electroactive properties (e-biochar) can be an ideal candidate for this scope, being at the same time widely available, biocompatible, and fully recyclable at its end-of-life as a soil amendment. Here we review the application of biochar to MET, to set benchmarks aimed at tuning the electroactive properties of such materials from the point of view of MET. The precursor biomass, thermochemical process conditions, and pre-, in situ-, and/or post-treatments should tailor optimized combinations of electrical conductivity, capacitance, superficial redox-active and electroactive functional groups, porosity distribution, and capacity to host electroactive microbial communities. We also discuss methods to rigorously characterize e-biochar properties and the most relevant multidisciplinary research challenges toward its application in large-scale MET. This work has been financed by the Italian Ministry of University and Research (MIUR), within the SIR2014 Grant, project RBSI14JKU3. Dr. R. Berenguer also thanks the Spanish Ministerio de Economía y Competitividad and FEDER funds (RYC-2017-23618 and CTM2015-71520-C2-1-R) for financial support. Ricardo Louro and Catarina Paquete thank Fundação para a Ciência e a Tecnologia (FCT) Portugal [PTDC/BBBBQB/4178/2014 and PTDC/BIA-BQM/30176/2017], by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI), and by ITQB research unit GREEN-it “Bioresources for sustainability” (UID/Multi/04551/2013). This work has also received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 810856. This investigation has also received funding from the European Union’s Horizon 2020 research and innovation programme under the grant agreement No. 642190 (Project “iMETLAND”; http://www.imetland.eu).

Crude glycerol is an undesired by-product of biodiesel production with a low commercial value (i.e. a ton of biodiesel results in around 110 kg of crude glycerol) and, thus, glycerol needs valorization. In particular, there is a need of providing a benefit to alkaline wastewaters from biodiesel production with excess of glycerol. Bioelectrochemical systems (BES) are an emerging technique to recover the energy contained in a substrate either as electricity or as other added-value products such as hydrogen. Moreover, promising results have been reported with alkaline BES showing higher current intensities than neutral pH conditions. This study is the first experimental evaluation of alkaline bioelectrochemical production of hydrogen from real crude glycerol as sole carbon source. The results show that alkaline glycerol degradation is feasible under both microbial fuel cell mode (2 mA, 71.4 A/m3 and 55% of CE) and microbial electrolysis mode (maximum of 0.46 LH2/L/d and 85% of rCAT). The values obtained are promising since they are in the range of those obtained with other simpler carbon sources such acetate. A complex consortium involving fermentative bacteria (such as Enterococcaceae), alkaline exoelectrogens (such as Geoalkalibacter) and homoacetogens (such as Acetobacterium) was naturally developed in the anode of the MEC.

The aim of this work were to study terracotta-based porous air-water separators (4 mm thickness) in microbial recycling cells (MRCs) fed with cow manure (CM), swine manure (SM) and dairy wastewater (DW). Over 125 days, besides the removal of 60–90% of soluble-COD, considerable fractions of the main macronutrients (C, N, P, K, Fe, Mn, Ca, Mg) were removed from the wastewater and deposited on the terracotta separators as both inorganic salts and biomass deposits. Water evaporation at air-water interface as well as the high cathodic pH (10–12), induced by oxygen reduction to OH–, were the predominant factors leading to precipitation. The separators were saturated of up to 10 g per kg of terracotta of the main macronutrients, with negligible concentrations of the main inorganic contaminants. These materials could be directly reused as nutrients-enriched solid conditioners for agricultural soils.

An anodic syntrophic consortium (exoelectrogenic plus fermentative bacteria) able to use methanol as sole carbon source was developed for the first time in a bioelectrochemical system. In this frame, promising results were obtained in single chamber MFC, comparable to those obtained with readily biodegradable substrates. Regarding MEC operation, the presence of homoacetogenic bacteria led to electron recycling, avoiding net hydrogen production in single chamber MEC. In a double chamber MEC, satisfying results (in terms of coulombic efficiency and cathodic gas recovery) were obtained even though energy recovery still restrained the feasibility of the process. The approach used in this work with methanol opens a new range of possibilities for other complex substrates as electron donors for bioelectrosynthesis.

Geobacter sulfurreducens is the model for electroactive microorganisms (EAM). EAM can use solid state terminal electron acceptors (TEA) including anodes via extracellular electron transfer (EET). Yield coefficients relate the produced cell number or biomass to the oxidized substrate or the reduced TEA. These data are not yet sufficiently available for EAM growing at anodes. Thus, this study provides information about kinetics as well as yield coefficients of early-stage G. sulfurreducens biofilms using anodes as TEA at the potentials of −200 mV, 0 mV and +200 mV (vs. Ag/AgCl sat. KCl). The selected microorganism was therefore cultivated in single and double chamber batch reactors on graphite or AuPd anodes. Interestingly, whereas the lag time and maximum current density within 12 days of growth differed, the anode potential does not influence the coulombic efficiency and the formal potential of the EET, which remains constant for all the experiments at ~ −300 to −350 mV. We demonstrated for the first time that the anode potential has a strong influence on single cell yield coefficients which ranged from 2.69 × 1012 cells mole-−1 at −200 mV and 1.48 × 1012 cells mole-−1 at 0 mV to 2.58 × 1011 cells mole-−1 at +200 mV in single chamber reactors and from 1.15 × 1012 cells mole-−1 at −200 mV to 8.98× 1011 cells mole-−1 at 0 mV in double chamber reactors. This data can be useful for optimization and scaling-up of primary microbial electrochemical technologies.

Recently, terracotta has attracted interest as low-cost and biocompatible material to build separators in microbial fuel cells (MFCs). However, the influence of a non-conductive material like terracotta on electroactive microbiological communities remains substantially unexplored. This study aims at describing the microbial pools developed from two different seed inocula (bovine and swine sewage) in terracotta-based air-breathing MFC. A statistical approach on microbiological data confirmed different community enrichment in the MFCs, depending mainly on the inoculum. Terracotta separators impeded the growth of electroactive communities in contact with cathodes (biocathodes), while a thick biofilm was observed on the surface (anolyte-side) of the terracotta separator. Terracotta-free MFCs, set as control experiments, showed a well-developed biocathode, Biocathode-MFCs resulted in 4 to 6-fold higher power densities. All biofilms were analyzed by high-throughput Illumina sequencing applied to 16S rRNA gene. The results showed more abundant (3- to 5-fold) electroactive genera (mainly Geobacter, Pseudomonas, Desulfuromonas and Clostridia MBA03) in terracotta-free biocathodes. Nevertheless, terracotta separators induced only slight changes in anodic microbial communities.

Dissolved oxygen (DO) at cathodic interface is a critical factor influencing microbial fuel cells (MFC) performance. In this work, three MFCs were operated with cathode under different DO conditions: i) air-breathing (A-MFC); ii) water-submerged (W-MFC) and iii) assisted by photosynthetic microorganisms (P-MFC). A plateau of maximum current was reached at 1.06±0.03mA, 1.48±0.06mA and 1.66±0.04mA, increasing respectively for W-MFC, P-MFC and A-MFC. Electrochemical and microbiological tools (Illumina sequencing, confocal microscopy and biofilm cryosectioning) were used to explore anodic and cathodic biofilm in each MFC type. In all cases, biocathodes improved oxygen reduction reaction (ORR) as compared to abiotic condition and A-MFC was the best performing system. Photosynthetic cultures in the cathodic chamber supplied high DO level, up to 16mgO2L-1, which sustained aerobic microbial community in P-MFC biocathode. Halomonas, Pseudomonas and other microaerophilic genera reached >50% of the total OTUs. The presence of sulfur reducing bacteria (Desulfuromonas) and purple non-sulfur bacteria in A-MFC biocathode suggested that the recirculation of sulfur compounds could shuttle electrons to sustain the reduction of oxygen as final electron acceptor. The low DO concentration limited the cathode in W-MFC. A model of two different possible microbial mechanisms is proposed which can drive predominantly cathodic ORR.

Microbial electroactivity, the metabolically relevant transfer of electrons between microorganisms and solid conductors, was first discovered for now well characterized model organisms from hypoxic or anaerobic water or sediment samples. Recent findings indicate that the metabolic trait of electroactivity might as well be important within the microbiome of the mammalian gut. Based on a pre-selection from the mouse intestinal bacterial collection five microorganisms originating from diverse parts of the gut were screened for electroactivity. As there is no marker gene for electroactivity, the ability to synthesize cytochromes and metabolize redox-mediators was studied in-silico. Clostridium cochlearium showed highest electroactivity and Lactobacillus reuteri as well as Staphylococcus xylosus show putative electroactivity, as well. The maximum current density of C. cochlearium of 0.53 ± 0.02 mA cm−2 after only 5.2 h of incubation was clearly linked to growth and glucose consumption. Cyclic voltammetric analysis on C. cochlearium revealed a formal potential of the extracellular electron transfer (EET) site of +0.22 ± 0.05 V versus Ag/AgCl sat. KCl (and + 0.42 V versus SHE) and indicates that EET is not based on biofilm formation, but the involvement of either redox-active molecules or planktonic cells. The potential of the gut as habitat for electroactives and their physiological role are discussed.

The gut microbiome is not only an indicator of different pathologies, but it also influences metabolism and overall health of the host. Recently, microorganisms inherent to the gut microbiome, such as Listeria monocytogenes, Enterococcus faecalis and Clostridium cochlearium, were demonstrated to be electroactive, i.e. to perform extracellular electron transfer (EET). To further explore the presence of electroactive microorganisms in the gut microbiome electrochemical enrichment starting from mouse feces was performed. Open circuit, abiotic and autoclaved inoculum controls were run in parallel. A maximum current density of 122±23 µA cm−2 at low coulombic efficiency ( 1%) was achieved. The presence of biofilms at the anode and microbial electrochemical activity with a formal potential of EET of 0.23±0.01 V vs. Ag/AgCl sat. KCl was demonstrated using fluorescence microscopy and cyclic voltammetry. The 16S rRNA gene sequencing and PCR-free Nanopore sequencing showed the enrichment and dominance of Shigella flexneri.

This work reports the first successful enrichment and operation of alkaline bioelectrochemical systems (microbial fuel cells, MFC, and microbial electrolysis cells, MEC). Alkaline (pH=9.3) bioelectrochemical hydrogen production presented better performance (+117%) compared to conventional neutral conditions (2.6 vs 1.2 litres of hydrogen gas per litre of reactor per day, LH2·L(-1)REACTOR·d(-1)). Pyrosequencing results of the anodic biofilm showed that while Geobacter was mainly detected under conventional neutral conditions, Geoalkalibacter sp. was highly detected in the alkaline MFC (21%) and MEC (48%). This is the first report of a high enrichment of Geoalkalibacter from an anaerobic mixed culture using alkaline conditions in an MEC. Moreover, Alkalibacter sp. was highly present in the anodic biofilm of the alkaline MFC (37%), which would indicate its potentiality as a new exoelectrogen.

BACKGROUND Microbial electrochemical systems (MXCs) are an emerging technology aiming at recovering energy contained in wastewaters either as electrical energy in microbial fuel cells (MFCs) or as hydrogen in microbial electrolysis cells (MECs). Successful results have been reported with readily biodegradable substrates, but the performance with real complex substrates needs to be evaluated to bridge the gap between lab- and full-scale. This work aims at studying bioelectrochemical hydrogen production using real cheese whey as sole substrate. RESULTS A microbial consortium able to consume cheese whey to produce electricity or H2 was developed. Cheese whey was fermented mainly by lactic acid bacteria (Enterococcus genus) and exoelectrogenic activity was performed by Geobacter sp. The coulombic efficiency was 49 ± 8% in the MFC fed only with cheese whey, which is higher than most previous values reported for MFCs fed with dairy products. Good results for H2 production in MEC (0.8 LH2 L−1REACTOR d−1) were also obtained. CONCLUSION The high potentiality of cheese whey as carbon source for bioelectrochemical systems is demonstrated in this study. The populations involved were determined by advanced microbial tools. The efficient selection of a syntrophic consortium to produce H2 directly from cheese whey in a single-chamber MEC was demonstrated. © 2016 Society of Chemical Industry

The use of synthetic wastewater containing carbon sources of different complexity (glycerol, milk and starch) was evaluated in single chamber microbial electrolysis cell (MEC) for hydrogen production. The growth of an anodic syntrophic consortium between fermentative and anode respiring bacteria was operationally enhanced and increased the opportunities of these complex substrates to be treated with this technology. During inoculation, current intensities achieved in single chamber microbial fuel cells were 50, 62.5, and 9 A m −3 for glycerol, milk and starch respectively. Both current intensities and coulombic efficiencies were higher than other values reported in previous works. The simultaneous degradation of the three complex substrates favored power production and COD removal. After three months in MEC operation, hydrogen production was only sustained with milk as a single substrate and with the simultaneous degradation of the three substrates. The later had the best results in terms of current intensity (150 A m −3 ), hydrogen production (0.94 m 3 m −3 d −1 ) and cathodic gas recovery (91%) at an applied voltage of 0.8 V. Glycerol and starch as substrates in MEC could not avoid the complete proliferation of hydrogen scavengers, even under low hydrogen retention time conditions induced by continuous nitrogen sparging.

The industrial synthesis (Haber-Bosch process) of mineral-N has been showing its dark side. Facing the enormous challenge of feeding humankind new technological solutions should enable sustainable nitrogen (N) fixation, to preserve soil fertility. We introduce a new type of microbial electrosynthesis, aimed at simultaneously fixing N2 and inorganic carbon. In this new perspective, the aim is enhancing biological nitrogen fixation by electrostimulation of complex microbial communities, to produce whole biomass, which can be used as soil fertilizer. Two preliminary contributions in this field reported: a) a bioelectrochemical version of the Haber-bosh process, where methyl viologen shuttled electrons to purified nitrogenase enzymes (Milton et al., 2017) and b) the stimulation of N-fixation in a pure planktonic culture of X. autotrophicus by H2-evolution with a specific abiotic catalyst (Liu et al., 2017). Here, a cathodic biofilm was enriched in electro-active autotrophic nitrogen fixers, starting from a mixed microbial culture. The cathode, a carbon-fibers conductor, was kept under constant polarization (-0.7 V vs SHE). Biomass (in the biofilm, as well as in the bulk liquid phase) was synthesized at significantly higher rates (up to 18-fold), as compared to controls kept at open circuit (OC). Along over 100 days, electron transfer had increased by 30-fold, as compared to abiotic conditions. Metagenomics evidenced Nif genes associated to autotrophs (both Archaea and Bacteria) only in polarized biofilms, while not in OC control. Genes copies encoding for a series of known proteins associated to extracellular electron transfer were double in polarized trials, as compared to controls. The first results of such an approach were recently published in Bioelectrochemistry (Rago et al., 2019). From this proof-of-the-concept, we propose to call this promising field ‘bio-electrochemical nitrogen fixation (e-BNF)’, that deserve future research efforts in both fundamentals and applicative aspects. Liu, C., Sakimoto, K.K., Colón, B.C., Silver, P.A., Nocera, D.G., 2017. Ambient nitrogen reduction cycle using a hybrid inorganic–biological system. Proc. Natl. Acad. Sci. 114, 6450–6455. doi:10.1073/pnas.1706371114 Milton, R.D., Cai, R., Abdellaoui, S., Leech, D., De Lacey, A.L., Pita, M., Minteer, S.D., 2017. Bioelectrochemical Haber–Bosch Process: An Ammonia-Producing H2/N2Fuel Cell. Angew. Chemie - Int. Ed. 56, 2680–2683. doi:10.1002/anie.201612500 Rago, L., Zecchin, S., Villa, F., Goglio, A., Corsini, A., Cavalca, L. & Schievano, A. 2019, ‘Bioelectrochemical Nitrogen fixation (e-BNF): Electro-stimulation of enriched biofilm communities drives autotrophic nitrogen and carbon fixation’, Bioelectrochemistry, vol. 125, pp. 105–15. Figure 1

BACKGROUND The microbial fuel cell (MFC) technology transforms the chemical energy present in substrates into electricity. Starting-up these systems, i.e. enriching the anodic community in exoelectrogenic bacteria, is a lengthy process or requires expensive equipment. RESULTS An easy and low-cost procedure based on a sediment MFC was developed to select microbial communities with exoelectrogenic activity from the anaerobic sludge of a waste water treatment plant (WWTP). The configuration was based on a simple vessel working as a single chamber MFC with a cathode of stainless steel wool in the liquid surface and a submerged graphite fibre brush as anode. In 30 days of operation, a biofilm with remarkable exoelectrogenic activity was grown on the anode of the MFC. This graphite fibre brush anode was able to supply 0.9 W m-2 when working in an air-cathode MFC (AC-MFC) for 45 days. CONCLUSION The procedure presented was demonstrated to be a successful, low-cost and low-maintenance procedure to obtain exoelectrogenic activity and had performances comparable with other more costly and complex inoculation procedures. The Sed-MFC does not require a potentiostat, external aeration, stirring, membranes or an enriched inoculum in the exoelectrogenic biomass. © 2013 Society of Chemical Industry

Bioelectrochemical systems need an anode with a high abundance of exoelectrogenic bacteria for an optimal performance. Among all possible operational parameters for an efficient enrichment, the role of external resistance in microbial fuel cell (MFC) has gained a lot of interest since it indirectly poises an anode potential, a key parameter for biofilm distribution and morphology. Thus, this work aims at investigating and discussing whether bioanodes selected at different external resistances under MFC operation present different responses under both MFC and microbial electrolysis cell (MEC) operation. A better MEC performance (i.e. shorter start-up time, higher current intensity and higher H2 production rate) was obtained with an anode from an MFC developed under low external resistance. Quantitative real-time polymerase chain reaction (qPCR) confirmed that a low external resistance provides an MFC anodic biofilm with the highest content of Geobacter because it allows higher current intensity, which is correlated to exoelectrogenic activity. High external resistances such as 1,000 Ω led to a slower start-up time under MEC operation.

Thriving under alkaliphilic conditions, Geoalkalibacter ferrihydriticus (Glk. ferrihydriticus) provides new applications in treating alkaline waste streams as well as a possible new model organism for microbial electrochemistry. We investigated the electrochemical response of biofilms of the alkaliphilic anode-respiring bacterium (ARB) Glk. ferrihydriticus voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronoamperometry. We observed there to be at least four dominant electron transfer pathways, with their contribution to the overall current produced dependent on the set anode potential. These pathways appear to be manifested at midpoint potentials of approximately -0.14 V, -0.2 V, -0.24 V, and -0.27 V vs standard hydrogen electrode. The individual contributions of the pathways change upon equilibration from a set anode potential to another anode potential. Additionally, the contribution of each pathway to the overall current produced is reversible when the anode potential is changed back to the original set potential. The pathways involved in anode respiration in Glk. ferrihydriticus biofilms follow a similar, but more complicated, pattern as compared to those in the model ARB, Geobacter sulfurreducens. This greater diversity of electron transport pathways in Glk. ferrihydriticus could be related to its wider metabolic capability (e.g., higher pH and larger set of possible substrates, among others).

A single-chamber microbial electrolysis cell (MEC) aiming at hydrogen production with acetate as sole carbon source failed due to methanogenesis build-up despite the significant amount of 2-bromoethanesulfonate (BES) dosage, 50 mM. Specific batch experiments and a thorough microbial community analysis, pyrosequencing and qPCR, of cathode, anode and medium were performed to understand these observations. The experimental data rebuts different hypothesis and shows that methanogenesis at high BES concentration was likely due to the capacity of some Archaea (hydrogen-oxidizing genus Methanobrevibacter) to resist high BES concentration up to 200 mM. Methanobrevibacter, of the Methanobacteriales order, represented almost the 98% of the total Archaea in the cathode whereas Geobacter was highly abundant in the anode (72% of bacteria). Moreover, at higher BES concentration (up to 200 mM), methanogenesis activity decreased resulting in an increase of homoacetogenic activity, which challenged the performance of the MEC for H2 production.

2-Bromoethanesulfonate (BES) is the most reported chemical inhibitor for methanogenesis in laboratory-scale bioelectrochemical systems. However, there is doubt about BES's long-term effectiveness in microbial fuel cells (MFCs). We observed BES degradation in MFCs, whereas not in microbial electrolysis cells (MECs). Our results suggest that BES degradation is only possible under aerobic conditions (such as in MFCs) when some oxygen diffuses through the cathode. Experimental BES degradation was linked to the release of bromide (Br−) into the medium, with an average recovery of 67 ± 16%. Microbial analysis of the cathodic biomass distribution revealed the presence of Pseudomonas and Alcaligenes genera, which are able to use sulfonates as carbon or sulfur sources under aerobic conditions.

Pall rings, a common random packing material, were used in the biotrickling filtration of biogas with high H2S. Assessment of 600d of operation covered the reactor start-up, the operation at neutral pH and the transition from neutral to acid pH. During the start-up period, operational parameters such as the aeration rate and the trickling liquid velocity were optimized. During the steady-state operation at neutral pH, the performance of the random packing material was investigated by reducing the gas contact time at both constant and increasing H2S loads. The random packing material showed similar elimination capacities and removal efficiencies in comparison with previous studies with a structured packing material, indicating that Pall rings are suitable for biogas desulfurization in biotrickling filters. The diversity of Eubacteria and the structure of the community were investigated before and after the pH transition using the bacterial tag-encoded FLX amplicon pyrosequencing. The pH transition to acid pH drastically reduced the microbial diversity and produced a progressive specialization of the sulfur-oxidizing bacteria community without any detrimental effect on the overall desulfurizing capacity of the reactor. During acidic pH operation, a persistent accumulation of elemental sulfur was found.