How Comparable are Microbial Electrochemical Systems around the Globe? An Electrochemical and Microbiological Cross-Laboratory Study.

Invited for this month's cover is the collaborative work among Univ. of Milano-Bicocca, Ricerca sul Sistema Energetico S.p.A., Univ. degli Studi di Milano, Univ. of California Irvine, Univ. of New Mexico, CNRS Toulouse. Technische Univ. Braunschweig, Aquacycl LLC, J. Craig Venter Institute, Helmholtz-Centre for Environmental Research. The image shows a sketch of a microbial fuel cell and a target indicating the need of developing common standards for the field of microbial electrochemical technologies. The Full Paper itself is available at 10.1002/cssc.202100294.

How Comparable are Microbial Electrochemical Systems around the Globe? An Electrochemical and Microbiological Cross-Laboratory Study.

A cross-laboratory study on microbial fuel cells (MFC) which involved different institutions around the world is presented. The study aims to assess the development of autochthone microbial pools enriched from domestic wastewater, cultivated in identical single-chamber MFCs, operated in the same way, thereby approaching the idea of developing common standards for MFCs. The MFCs are inoculated with domestic wastewater in different geographic locations. The acclimation stage and, consequently, the startup time are longer or shorter depending on the inoculum, but all MFCs reach similar maximum power outputs (55±22 µW cm-2 ) and COD removal efficiencies (87±9 %), despite the diversity of the bacterial communities. It is inferred that the MFC performance starts when the syntrophic interaction of fermentative and electrogenic bacteria stabilizes under anaerobic conditions at the anode. The generated power is mostly limited by electrolytic conductivity, electrode overpotentials, and an unbalanced external resistance. The enriched microbial consortia, although composed of different bacterial groups, share similar functions both on the anode and the cathode of the different MFCs, resulting in similar electrochemical output.

Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium.

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c-type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.

Continuous flow, large-scale, microbial fuel cell system for the sustained treatment of swine waste.

Microbial fuel cells (MFCs) have long held the promise of being a cost-effective technology for the energy-neutral treatment of wastewater. However, successful pilot-scale demonstrations for this technology are still limited to very few. Here, we present a large-scale MFC system, composed of 12 MFCs with a total volume of 110 L, successfully treating swine wastewater at a small educational farm. The system was operated for over 200 days in continuous mode with hydraulic residence time of 4 hr. Very stable electrochemical and waste treatment performance was observed with up to 65% of chemical oxygen demand (COD) removed and a maximum treatment rate of 5.0 kg COD/m3 .day. Robust microbial enrichment was performed and adapted to metabolize and transform a diversity of compounds present. The Net Energy Recovery (NER = 0.11 kWhr/kg COD) is not only competitive with conventional cogeneration processes, but is in fact sufficient to sustain the operational energy requirements of the system. PRACTITIONER POINTS: This study demonstrates the design and operation of a large-scale microbial fuel cells (MFC) system for continuous treatment of swine wastewater. The system achieved a high chemical oxygen demand removal rate within a short hydraulic residence time. This study moves one-step closer to applying MFC technology for real wastewater treatment.

Comparative metatranscriptomics reveals extracellular electron transfer pathways conferring microbial adaptivity to surface redox potential changes.

Some microbes can capture energy through redox reactions with electron flow to solid-phase electron acceptors, such as metal-oxides or poised electrodes, via extracellular electron transfer (EET). While diverse oxide minerals, exhibiting different surface redox potentials, are widely distributed on Earth, little is known about how microbes sense and use the minerals. Here we show electrochemical, metabolic, and transcriptional responses of EET-active microbial communities established on poised electrodes to changes in the surface redox potentials (as electron acceptors) and surrounding substrates (as electron donors). Combination of genome-centric stimulus-induced metatranscriptomics and metabolic pathway investigation revealed that nine Geobacter/Pelobacter microbes performed EET activity differently according to their preferable surface potentials and substrates. While the Geobacter/Pelobacter microbes coded numerous numbers of multi-heme c-type cytochromes and conductive e-pili, wide variations in gene expression were seen in response to altering surrounding substrates and surface potentials, accelerating EET via poised electrode or limiting EET via an open circuit system. These flexible responses suggest that a wide variety of EET-active microbes utilizing diverse EET mechanisms may work together to provide such EET-active communities with an impressive ability to handle major changes in surface potential and carbon source availability.

Outer membrane cytochromes/flavin interactions in Shewanella spp.-A molecular perspective.

Extracellular electron transfer (EET) is intrinsically associated with the core phenomena of energy harvesting/energy conversion in natural ecosystems and biotechnology applications. However, the mechanisms associated with EET are complex and involve molecular interactions that take place at the "bionano interface" where biotic/abiotic interactions are usually explored. This work provides molecular perspective on the electron transfer mechanism(s) employed by Shewanella oneidensis MR-1. Molecular docking simulations were used to explain the interfacial relationships between two outer-membrane cytochromes (OMC) OmcA and MtrC and riboflavin (RF) and flavin mononucleotide (FMN), respectively. OMC-flavin interactions were analyzed by studying the electrostatic potential, the hydrophilic/hydrophobic surface properties, and the van der Waals surface of the OMC proteins. As a result, it was proposed that the interactions between flavins and OMCs are based on geometrical recognition event. The possible docking positions of RF and FMN to OmcA and MtrC were also shown.

Population dynamics of electrogenic microbial communities in microbial fuel cells started with three different inoculum sources.

Microbial fuel cells (MFCs) are one of the bioelectrochemical systems that exploit microorganisms as biocatalysts to degrade organic matters and recover energy as electric power. Here, we explored how the established electrogenic microbial communities were influenced by three different inoculum sources; anaerobic sludge of the wastewater plant, rice paddy field soil, and coastal lagoon sediment. We periodically characterized both electricity generation with sucrose consumption and 16S rRNA-basis microbial community composition. The electrochemical features of MFCs were slightly different among three inocula, and the lagoon sediment-inoculated MFC showed the highest performance in terms of the treatment time. Meanwhile, although the inoculated microbial communities were highly diverse and quite different, only twelve genera affiliated with δ-Proteobacteria, γ-Proteobacteria, Bacilli, Clostridia/Negativicutes or Bacteroidetes were abundantly enriched in all MFC anode communities. Within them, several fermentative genera were clearly different due to the inocula, while the inocula-specific phylotypes were identified in an electrogenic genus Geobacter. The relative abundances of phylotypes closely-related to Geobacter metallireducens were increased in later stages of all the sucrose-fed MFCs. These results indicate that key microbial members for the functional electrogenic community widely exist in natural ecosystems, but the community members presenting in inoculum sources affected the MFC performances.

Harnessing the Power of PCR Molecular Fingerprinting Methods and Next Generation Sequencing for Understanding Structure and Function in Microbial Communities.

Polymerase chain reaction (PCR) is central to methods in molecular ecology. Here, we describe PCR-dependent approaches useful for investigating microbial diversity and its function in various natural, human-associated, and built environment ecosystems. Protocols routinely used for DNA extraction, purification, cloning, and sequencing are included along with various resources for the statistical analysis following gel electrophoresis-based methods (DGGE) and sequencing. We also provide insights into eukaryotic microbiome analysis, sample preservation techniques, PCR troubleshooting, DNA quantification methods, and commonly used ordination techniques.

Effect of oxygen on the per-cell extracellular electron transfer rate of Shewanella oneidensis MR-1 explored in bioelectrochemical systems.

Extracellular electron transfer (EET) is a mechanism that enables microbes to respire solid-phase electron acceptors. These EET reactions most often occur in the absence of oxygen, since oxygen can act as a competitive electron acceptor for many facultative microbes. However, for Shewanella oneidensis MR-1, oxygen may increase biomass development, which could result in an overall increase in EET activity. Here, we studied the effect of oxygen on S. oneidensis MR-1 EET rates using bioelectrochemical systems (BESs). We utilized optically accessible BESs to monitor real-time biomass growth, and studied the per-cell EET rate as a function of oxygen and riboflavin concentrations in BESs of different design and operational conditions. Our results show that oxygen exposure promotes biomass development on the electrode, but significantly impairs per-cell EET rates even though current production does not always decrease with oxygen exposure. Additionally, our results indicated that oxygen can affect the role of riboflavin in EET. Under anaerobic conditions, both current density and per-cell EET rate increase with the riboflavin concentration. However, as the dissolved oxygen (DO) value increased to 0.42 mg/L, riboflavin showed very limited enhancement on per-cell EET rate and current generation. Since it is known that oxygen can promote flavins secretion in S. oneidensis, the role of riboflavin may change under anaerobic and aerobic conditions. Biotechnol. Bioeng. 2017;114: 96-105. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Co-generation of hydrogen and power/current pulses from supercapacitive MFCs using novel HER iron-based catalysts.

In this work, four different supercapacitive microbial fuel cells (SC-MFCs) with carbon brush as the anode and an air-breathing cathode with Fe-Aminoantipyrine (Fe-AAPyr) as the catalyst have been investigated using galvanostatic discharges. The maximum power (Pmax) obtained was in the range from 1.7 mW to 1.9 mW for each SC-MFC. This in-series connection of four SC-MFCs almost quadrupled Pmax to an operating voltage of 3025 mV and a Pmax of 8.1 mW, one of the highest power outputs reported in the literature. An additional electrode (AdHER) connected to the anode of the first SC-MFC and placed in the fourth SC-MFC evolved hydrogen. The hydrogen evolution reaction (HER) taking place at the electrode was studied on Pt and two novel platinum group metal-free (PGM-free) catalysts: Fe-Aminoantipyrine (Fe-AAPyr) and Fe-Mebendazole (Fe-MBZ). The amount of H2 produced was estimated using the Faraday law as 0.86 mMd-1cm-2 (0.132 L day-1) for Pt, 0.83 mMd-1cm-2 (0.127 L day-1) for Fe-AAPyr and 0.8 mMd-1cm-2 (0.123 L day-1) for Fe-MBZ. Hydrogen evolution was also detected using gas chromatography. While HER was taking place, galvanostatic discharges were also performed showing simultaneous H2 production and pulsed power generation with no need of external power sources.

Microbial metabolic networks in a complex electrogenic biofilm recovered from a stimulus-induced metatranscriptomics approach.

Microorganisms almost always exist as mixed communities in nature. While the significance of microbial community activities is well appreciated, a thorough understanding about how microbial communities respond to environmental perturbations has not yet been achieved. Here we have used a combination of metagenomic, genome binning, and stimulus-induced metatranscriptomic approaches to estimate the metabolic network and stimuli-induced metabolic switches existing in a complex microbial biofilm that was producing electrical current via extracellular electron transfer (EET) to a solid electrode surface. Two stimuli were employed: to increase EET and to stop EET. An analysis of cell activity marker genes after stimuli exposure revealed that only two strains within eleven binned genomes had strong transcriptional responses to increased EET rates, with one responding positively and the other responding negatively. Potential metabolic switches between eleven dominant members were mainly observed for acetate, hydrogen, and ethanol metabolisms. These results have enabled the estimation of a multi-species metabolic network and the associated short-term responses to EET stimuli that induce changes to metabolic flow and cooperative or competitive microbial interactions. This systematic meta-omics approach represents a next step towards understanding complex microbial roles within a community and how community members respond to specific environmental stimuli.

Functional and taxonomic dynamics of an electricity-consuming methane-producing microbial community.

The functional and taxonomic microbial dynamics of duplicate electricity-consuming methanogenic communities were observed over a 6 months period to characterize the reproducibility, stability and recovery of electromethanogenic consortia. The highest rate of methanogenesis was 0.72 mg-CH4/L/day, which occurred during the third month of enrichment when multiple methanogenic phylotypes and associated Desulfovibrionaceae phylotypes were present in the electrode-associated microbial community. Results also suggest that electromethanogenic microbial communities are very sensitive to electron donor-limiting open-circuit conditions. A 45 min exposure to open-circuit conditions induced an 87% drop in volumetric methane production rates. Methanogenic performance recovered after 4 months to a maximum value of 0.30 mg-CH4/L/day under set potential operation (-700 mV vs Ag/AgCl); however, current consumption and biomass production was variable over time. Long-term functional and taxonomic analyses from experimental replicates provide new knowledge toward understanding how to enrich electromethanogenic communities and operate bioelectrochemical systems for stable and reproducible performance.

Influence of anode surface chemistry on microbial fuel cell operation.

Self-assembled monolayers (SAMs) modified gold anodes are used in single chamber microbial fuel cells for organic removal and electricity generation. Hydrophilic (N(CH3)3(+), OH, COOH) and hydrophobic (CH3) SAMs are examined for their effect on bacterial attachment, current and power output. The different substratum chemistry affects the community composition of the electrochemically active biofilm formed and thus the current and power output. Of the four SAM-modified anodes tested, N(CH3)3(+) results in the shortest start up time (15 days), highest current achieved (225 µA cm(-2)) and highest MFC power density (40 µW cm(-2)), followed by COOH (150 µA cm(-2) and 37 µW cm(-2)) and OH (83 µA cm(-2) and 27 µW cm(-2)) SAMs. Hydrophobic SAM decreases electrochemically active bacteria attachment and anode performance in comparison to hydrophilic SAMs (CH3 modified anodes 7 µA cm(-2) anodic current and 1.2 µW cm(-2) MFC's power density). A consortium of Clostridia and δ-Proteobacteria is found on all the anode surfaces, suggesting a synergistic cooperation under anodic conditions.

Innovative statistical interpretation of Shewanella oneidensis microbial fuel cells data.

The last decade of research has made significant strides toward practical applications of Microbial Fuel Cells (MFCs); however, design improvements and operational optimization cannot be realized without equally considering engineering designs and biological interfacial reactions. In this study, the main factors contributing to MFCs' overall performance and their influence on MFC reproducibility are discussed. Two statistical approaches were used to create a map of MFC components and their expanded uncertainties, principal component analysis (PCA) and uncertainty of measurement results (UMR). PCA was used to identify the major factors influencing MFCs and to determine their ascendency over MFC operational characteristics statistically. UMR was applied to evaluate the factors' uncertainties and estimate their level of contribution to the final irreproducibility. In order to simplify the presentation and concentrate on the MFC components, only results from Shewanella spp. were included; however, a similar analysis could be applied for any DMRB or microbial community. The performed PCA/UMR analyses suggest that better reproducibility of MFC performance can be achieved through improved design parameters. This approach is exactly opposite to the MFC optimization and scale up approach, which should start with improving the bacteria-electrode interactions and applying these findings to well-designed systems.

Microbial population and functional dynamics associated with surface potential and carbon metabolism.

Microbial extracellular electron transfer (EET) to solid surfaces is an important reaction for metal reduction occurring in various anoxic environments. However, it is challenging to accurately characterize EET-active microbial communities and each member's contribution to EET reactions because of changes in composition and concentrations of electron donors and solid-phase acceptors. Here, we used bioelectrochemical systems to systematically evaluate the synergistic effects of carbon source and surface redox potential on EET-active microbial community development, metabolic networks and overall electron transfer rates. The results indicate that faster biocatalytic rates were observed under electropositive electrode surface potential conditions, and under fatty acid-fed conditions. Temporal 16S rRNA-based microbial community analyses showed that Geobacter phylotypes were highly diverse and apparently dependent on surface potentials. The well-known electrogenic microbes affiliated with the Geobacter metallireducens clade were associated with lower surface potentials and less current generation, whereas Geobacter subsurface clades 1 and 2 were associated with higher surface potentials and greater current generation. An association was also observed between specific fermentative phylotypes and Geobacter phylotypes at specific surface potentials. When sugars were present, Tolumonas and Aeromonas phylotypes were preferentially associated with lower surface potentials, whereas Lactococcus phylotypes were found to be closely associated with Geobacter subsurface clades 1 and 2 phylotypes under higher surface potential conditions. Collectively, these results suggest that surface potentials provide a strong selective pressure, at the species and strain level, for both solid surface respirators and fermentative microbes throughout the EET-active community development.

Identifying the microbial communities and operational conditions for optimized wastewater treatment in microbial fuel cells.

Microbial fuel cells (MFCs) are devices that exploit microorganisms as "biocatalysts" to recover energy from organic matter in the form of electricity. MFCs have been explored as possible energy neutral wastewater treatment systems; however, fundamental knowledge is still required about how MFC-associated microbial communities are affected by different operational conditions and can be optimized for accelerated wastewater treatment rates. In this study, we explored how electricity-generating microbial biofilms were established at MFC anodes and responded to three different operational conditions during wastewater treatment: 1) MFC operation using a 750 Ω external resistor (0.3 mA current production); 2) set-potential (SP) operation with the anode electrode potentiostatically controlled to +100 mV vs SHE (4.0 mA current production); and 3) open circuit (OC) operation (zero current generation). For all reactors, primary clarifier effluent collected from a municipal wastewater plant was used as the sole carbon and microbial source. Batch operation demonstrated nearly complete organic matter consumption after a residence time of 8-12 days for the MFC condition, 4-6 days for the SP condition, and 15-20 days for the OC condition. These results indicate that higher current generation accelerates organic matter degradation during MFC wastewater treatment. The microbial community analysis was conducted for the three reactors using 16S rRNA gene sequencing. Although the inoculated wastewater was dominated by members of Epsilonproteobacteria, Gammaproteobacteria, and Bacteroidetes species, the electricity-generating biofilms in MFC and SP reactors were dominated by Deltaproteobacteria and Bacteroidetes. Within Deltaproteobacteria, phylotypes classified to family Desulfobulbaceae and Geobacteraceae increased significantly under the SP condition with higher current generation; however those phylotypes were not found in the OC reactor. These analyses suggest that species related to family Desulfobulbaceae and Geobacteraceae are correlated with the electricity generation in the biofilm and may be key players for optimizing wastewater treatment rates and energy recovery in applied MFC systems.

Electricity generation by microbial fuel cell using microorganisms as catalyst in cathode.

The cathode reaction is one of the most seriously limiting factors in a microbial fuel cell (MFC). The critical dissolved oxygen (DO) concentration of a platinum-loaded graphite electrode was reported as 2.2 mg/l, about 10-fold higher than an aerobic bacterium. A series of MFCs were run with the cathode compartment inoculated with activated sludge (biotic) or not (abiotic) on platinum-loaded or bare graphite electrodes. At the beginning of the operation, the current values from MFCs with a biocathode and abiotic cathode were 2.3 ± 0.1 and 2.6 ± 0.2 mA, respectively, at the air-saturated water supply in the cathode. The current from MFCs with an abiotic cathode did not change, but that of MFCs with a biotic cathode increased to 3.0 mA after 8 weeks. The coulomb efficiency was 59.6% in the MFCs with a biotic cathode, much higher than the value of 15.6% of the abiotic cathode. When the DO supply was reduced, the current from MFCs with an abiotic cathode decreased more sharply than in those with a biotic cathode. When the respiratory inhibitor azide was added to the catholyte, the current decreased in MFCs with a biotic cathode but did not change in MFCs with an abiotic cathode. The power density was higher in MFCs with a biotic cathode (430 W/m(3) cathode compartment) than the abiotic cathode MFC (257 W/m(3) cathode compartment). Electron microscopic observation revealed nanowire structures in biofilms that developed on both the anode and on the biocathode. These results show that an electron consuming bacterial consortium can be used as a cathode catalyst to improve the cathode reaction.

A novel metatranscriptomic approach to identify gene expression dynamics during extracellular electron transfer.

Microbial respiration via extracellular electron transfer (EET) is a ubiquitous reaction that occurs throughout anoxic environments and is a driving force behind global biogeochemical cycling of metals. Here we identify specific EET-active microbes and genes in a diverse biofilm using an innovative approach to analyse the dynamic community-wide response to changing EET rates. We find that the most significant gene expression responses to applied EET stimuli occur in only two microbial groups, Desulfobulbaceae and Desulfuromonadales. Metagenomic analyses reveal high coverage draft genomes of these abundant and active microbes. Our metatranscriptomic results show known and unknown genes that are highly responsive to EET stimuli and associated with our identified draft genomes. This new approach yields a comprehensive image of functional microbes and genes related to EET activity in a diverse community, representing the next step towards unravelling complex microbial roles within a community and how microbes adapt to specific environmental stimuli.

Adaptation of soil microbes during establishment of microbial fuel cell consortium fed with lactate.

We report the development of microbial populations and changes in their electrochemical production during a 2-month study of a two-chamber microbial fuel cell (MFC). The original inoculum was taken from anaerobic enrichment cultures with soil as the inoculum, and lactate as the exogenous electron donor. Power density (PD), coulombic production (CP), and coulombic efficiency (CE) increased rapidly, reaching high values (320 mW m(-3), 65 Q, and 12.5%, respectively) in 12-16 days. Under these conditions, several major microbial taxa dominated the anode population. The medium solution in the cathode chamber decreased with aeration, resulting in a decrease in PD to 55 mW m(-3) at day 20. Refilling the cathode chamber around day 30 resulted in restoration of the PD, CP and CE to values equal to or greater than those previously observed. However, after the change in conditions, a marked change in community structure was observed, and high levels of acetate were seen in the anode chamber of the fuel cell for the first time. At day 35, a series of lactate concentrations were used, beginning with low levels and increasing to the 20 mM level originally used (day 46), the PD decreased but was stable at 150 mW m(-3) and the acetate concentration in the anode stabilized at about 35 mM. Under these conditions, new major population structures, which were closely related to Propionibacterium, Clostridium, and uncultured bacteria, were observed in the anode. These results suggested that the flexibility of community structure was important for sustainable electricity production.

Functionally stable and phylogenetically diverse microbial enrichments from microbial fuel cells during wastewater treatment.

Microbial fuel cells (MFCs) are devices that exploit microorganisms as biocatalysts to recover energy from organic matter in the form of electricity. One of the goals of MFC research is to develop the technology for cost-effective wastewater treatment. However, before practical MFC applications are implemented it is important to gain fundamental knowledge about long-term system performance, reproducibility, and the formation and maintenance of functionally-stable microbial communities. Here we report findings from a MFC operated for over 300 days using only primary clarifier effluent collected from a municipal wastewater treatment plant as the microbial resource and substrate. The system was operated in a repeat-batch mode, where the reactor solution was replaced once every two weeks with new primary effluent that consisted of different microbial and chemical compositions with every batch exchange. The turbidity of the primary clarifier effluent solution notably decreased, and 97% of biological oxygen demand (BOD) was removed after an 8-13 day residence time for each batch cycle. On average, the limiting current density was 1000 mA/m(2), the maximum power density was 13 mW/m(2), and coulombic efficiency was 25%. Interestingly, the electrochemical performance and BOD removal rates were very reproducible throughout MFC operation regardless of the sample variability associated with each wastewater exchange. While MFC performance was very reproducible, the phylogenetic analyses of anode-associated electricity-generating biofilms showed that the microbial populations temporally fluctuated and maintained a high biodiversity throughout the year-long experiment. These results suggest that MFC communities are both self-selecting and self-optimizing, thereby able to develop and maintain functional stability regardless of fluctuations in carbon source(s) and regular introduction of microbial competitors. These results contribute significantly toward the practical application of MFC systems for long-term wastewater treatment as well as demonstrating MFC technology as a useful device to enrich for functionally stable microbial populations.