Mechanisms of extracellular electron transfer in anaerobic methanotrophic archaea.

Anaerobic methanotrophic (ANME) archaea are environmentally important, uncultivated microorganisms that oxidize the potent greenhouse gas methane. During methane oxidation, ANME archaea engage in extracellular electron transfer (EET) with other microbes, metal oxides, and electrodes through unclear mechanisms. Here, we cultivate ANME-2d archaea ('Ca. Methanoperedens') in bioelectrochemical systems and observe strong methane-dependent current (91-93% of total current) associated with high enrichment of 'Ca. Methanoperedens' on the anode (up to 82% of the community), as determined by metagenomics and transmission electron microscopy. Electrochemical and metatranscriptomic analyses suggest that the EET mechanism is similar at various electrode potentials, with the possible involvement of an uncharacterized short-range electron transport protein complex and OmcZ nanowires.

Comparing theoretical and practical biomass yields calls for revisiting thermodynamic growth models for electroactive microorganisms.

Research on electroactive microorganisms (EAM) often focuses either on their physiology and the underlying mechanisms of extracellular electron transfer or on their application in microbial electrochemical technologies (MET). Thermodynamic understanding of energy conversions related to growth and activity of EAM has received only a little attention. In this study, we aimed to prove the hypothesized restricted energy harvest of EAM by determining biomass yields by monitoring growth of acetate-fed biofilms presumably enriched in Geobacter, using optical coherence tomography, at three anode potentials and four acetate concentrations. Experiments were concurrently simulated using a refined thermodynamic model for EAM. Neither clear correlations were observed between biomass yield and anode potential nor acetate concentration, albeit the statistical significances are limited, mainly due to the observed experimental variances. The experimental biomass yield based on acetate consumption (YX/ac = 37 ± 9 mgCODbiomass gCODac-1) was higher than estimated by modeling, indicating limitations of existing growth models to predict yields of EAM. In contrast, the modeled biomass yield based on catabolic energy harvest was higher than the biomass yield from experimental data (YX/cat = 25.9 ± 6.8 mgCODbiomass kJ-1), supporting restricted energy harvest of EAM and indicating a role of not considered energy sinks. This calls for an adjusted growth model for EAM, including, e.g., the microbial electrochemical Peltier heat to improve the understanding and modeling of their energy metabolism. Furthermore, the reported biomass yields are important parameters to design strategies for influencing the interactions between EAM and other microorganisms and allowing more realistic feasibility assessments of MET.

Starvation combined with constant anode potential triggers intracellular electron storage in electro-active biofilms.

The accumulation of electrons in the form of Extracellular Polymeric Substances (EPS) and poly-hydroxyalkanoates (PHA) has been studied in anaerobic processes by adjusting the access of microorganisms to the electron donor and final electron acceptor. In Bio-electrochemical systems (BESs), intermittent anode potential regimes have also recently been used to study electron storage in anodic electro-active biofilms (EABfs), but the effect of electron donor feeding mode on electron storage has not been explored. Therefore, in this study, the accumulation of electrons in the form of EPS and PHA was studied as a function of the operating conditions. EABfs were grown under both constant and intermittent anode potential regimes and fed with acetate (electron donor) continuously or in batch. Confocal Laser Scanning Microscopy (CLSM) and Fourier-Transform Infrared Spectroscopy (FTIR) were used to assess electron storage. The range of Coulombic efficiencies, from 25 to 82%, and the biomass yields, between 10 and 20%, indicate that storage could have been an alternative electron consuming process. From image processing, a 0.92 pixel ratio of poly-hydroxybutyrate (PHB) and amount of cells was found in the batch fed EABf grown under a constant anode potential. This storage was linked to the presence of living Geobacter and shows that energy gain and carbon source starvation were the triggers for intracellular electron storage. The highest EPS content (extracellular storage) was observed in the continuously fed EABf under an intermittent anode potential, showing that constant access to electron donor and intermittent access to the electron acceptor leads to the formation of EPS from the excess energy gained. Tailoring operating conditions can thus steer the microbial community and result in a trained EABf to perform a desired biological conversion, which can be beneficial for a more efficient and optimized BES.

Maximum thickness of non-buffer limited electro-active biofilms decreases at higher anode potentials.

The accumulation of protons in electro-active biofilms (EABfs) has been reported as a critical parameter determining produced currents at the anode since the very beginning of the studies on Bio-electrochemical systems (BESs). Even though the knowledge gained on the influence of this parameter on the produced currents, its influence on EABfs growth is frequently overlooked. In this study, we quantified EABfs thicknesses in real-time and related them to the produced current at three buffer concentrations, two anode potentials and two acetate concentrations. The thickest EABfs (80 µm) and higher produced currents (2.5 A.m-2) were measured when a 50 mM buffer concentration was used. By combining the measured EABfs thicknesses with the pH in the anolyte, a simple model was developed to identify buffer limitations. Buffer limited EABfs with thicknesses of 15 and 42 µm were identified at -0.3 V vs Ag/AgCl when 10 and 50 mM buffer concentrations were used, respectively. At -0.2 V vs Ag/AgCl, the thicknesses of buffer limited EABfs decreased to 13 and 20 µm, respectively. The model also estimated buffer and acetate diffusion rates in EABfs and allowed to determine the boundary between a buffer and acetate limited EABfs. The diffusion rates reported in this study and the definition of the boundary between buffer and acetate limited EABfs provide a powerful tool to avoid limitations, leading to higher produced currents at the anode.

Effects of Current on the Membrane and Boundary Layer Selectivity in Electrochemical Systems Designed for Nutrient Recovery.

During electrochemical nutrient recovery, current and ion exchange membranes (IEM) are used to extract an ionic species of interest (e.g., ion) from a mixture of multiple ions. The species of interest (ion 1) has an opposing charge to the IEM. When ion 1 is extracted from the solution, the species fractions at the membrane and the adjunct boundary layers are affected. Hence, the species transport through the electrochemical system (ES) can no longer be described as electrodialysis-like. A dynamic state is observed in the compartments, where the ionic species are recovered. When the boundary layer-membrane interface is depleted, the IEM is at maximum current. If the ES is operated at a current higher than the maximum current, the fluxes of both ion 1 and other competing ions, with the same charge (ion 2), occur. This means, for example, ion 1 will be recovered, and the concentration of ion 2 will build up in time. Therefore, a steady state is never reached. Ideally, to prevent the effect of limiting current at the boundary layer-membrane interface, ES for nutrient recovery should be operated at low currents.

Opportunities for visual techniques to determine characteristics and limitations of electro-active biofilms.

Optimization of bio-electrochemical systems (BESs) relies on a better understanding of electro-active biofilms (EABfs). These microbial communities are studied with a range of techniques, including electrochemical, visual and chemical techniques. Even though each of these techniques provides very valuable and wide-ranging information about EABfs, such as performance, morphology and biofilm composition, they are often destructive. Therefore, the information obtained from EABfs development and characterization studies are limited to a single characterization of EABfs and often limited to one time point that determines the end of the experiment. Despite being scarcer and not as commonly reported as destructive techniques, non-destructive visual techniques can be used to supplement EABfs characterization by adding in-situ information of EABfs functioning and its development throughout time. This opens the door to EABfs monitoring studies that can complement the information obtained with destructive techniques. In this review, we provide an overview of visual techniques and discuss the opportunities for combination with the established electrochemical techniques to study EABfs. By providing an overview of suitable visual techniques and discussing practical examples of combination of visual with electrochemical methods, this review aims at serving as a source of inspiration for future studies in the field of BESs.

Ammonia recovery from anaerobic digester centrate using onsite pilot scale bipolar membrane electrodialysis coupled to membrane stripping.

Ammonia recovery from centrate of an anaerobic digester was investigated using an onsite bipolar-electrodialysis (BP-ED) pilot scale plant coupled to two liquid/liquid membrane contactor (LLMC) modules. To investigate the process performance and robustness, the pilot plant was operated at varying current densities, load ratio (current to nitrogen loading), and in continuous and intermittent current (Donnan) mode. A higher load ratio led to higher total ammonium nitrogen (TAN, sum of ammonia and ammonium) removal efficiency, whereas the increase in the applied current did not have a significant impact the TAN removal efficiency. Continuous current application resulted in the higher TAN removal compared with the Donnan dialysis mode. The lowest specific energy consumption of 6.3 kWh kgN-1 was recorded in the Donnan mode, with the load ratio of 1.4, at 200 L h-1 flowrate and current density of 75 A m-2. Lower energy demand observed in the Donnan mode was likely due to the lower scaling and fouling of the ion exchange membranes. Nevertheless, scaling and fouling limited the operation of the BP-ED stack in all operational modes, which had to be interrupted by the daily cleaning procedures. The LLMC module enabled a highly selective recovery of ammonia as ammonium sulfate ((NH4)2SO4), with the concentration of ammonia ranging from 19 to 33 gN L-1. However, the analysis of per- and polyfluoroalkyl substances (PFASs) in the obtained (NH4)2SO4 product revealed the presence of 212-253 ng L-1 of 6:2 fluorotelomer sulfonate (FTS), a common substitute of legacy PFAS. Given the very low concentrations of 6:2 FTS (i.e., < 2 ng L-1) encountered in the concentrated stream, 6:2 FTS was likely released from the Teflon-based components in the sulfuric acid dosage line. Thus, careful selection of the pilot plant tubing, pumps and other components is required to avoid any risks associated with the PFAS presence and ensure safe use of the final product as fertilizer.

Methane-Dependent Extracellular Electron Transfer at the Bioanode by the Anaerobic Archaeal Methanotroph "Candidatus Methanoperedens".

Anaerobic methanotrophic (ANME) archaea have recently been reported to be capable of using insoluble extracellular electron acceptors via extracellular electron transfer (EET). In this study, we investigated EET by a microbial community dominated by "Candidatus Methanoperedens" archaea at the anode of a bioelectrochemical system (BES) poised at 0 V vs. standard hydrogen electrode (SHE), in this way measuring current as a direct proxy of EET by this community. After inoculation of the BES, the maximum current density was 274 mA m-2 (stable current up to 39 mA m-2). Concomitant conversion of 13CH4 into 13CO2 demonstrated that current production was methane-dependent, with 38% of the current attributed directly to methane supply. Based on the current production and methane uptake in a closed system, the Coulombic efficiency was about 17%. Polarization curves demonstrated that the current was limited by microbial activity at potentials above 0 V. The metatranscriptome of the inoculum was mined for the expression of c-type cytochromes potentially used for EET, which led to the identification of several multiheme c-type cytochrome-encoding genes among the most abundant transcripts in "Ca. Methanoperedens." Our study provides strong indications of EET in ANME archaea and describes a system in which ANME-mediated EET can be investigated under laboratory conditions, which provides new research opportunities for mechanistic studies and possibly the generation of axenic ANME cultures.

The effect of intermittent anode potential regimes on the morphology and extracellular matrix composition of electro-active bacteria.

Electro-active bacteria (EAB) can form biofilms on an anode (so-called bioanodes), and use the electrode as electron acceptor for oxidation of organics in wastewater. So far, bioanodes have mainly been investigated under a continuous anode potential, but intermittent anode potential has resulted in higher currents and different biofilm morphologies. However, little is known about how intermittent potential influences the electron balance in the anode compartment. In this study, we investigated electron balances of bioanodes at intermittent anode potential regimes. We used a transparent non-capacitive electrode that also allowed for in-situ quantification of the EAB using optical coherence tomography (OCT). We observed comparable current densities between continuous and intermittent bioanodes, and stored charge was similar for all the applied intermittent times (5 mC). Electron balances were further investigated by quantifying Extracellular Polymeric Substances (EPS), by analyzing the elemental composition of biomass, and by quantifying biofilm and planktonic cells. For all tested conditions, a charge balance of the anode compartment showed that more electrons were diverted to planktonic cells than biofilm. Besides, 27-43% of the total charge was detected as soluble EPS in intermittent bioanodes, whereas only 15% was found as soluble EPS in continuous bioanodes. The amount of proteins in the EPS of biofilms was higher for intermittent operated bioanodes (0.21 mg COD proteins mg COD biofilm-1) than for continuous operated bioanodes (0.05 mg COD proteins mg COD biofilm-1). OCT revealed patchy morphologies for biofilms under intermittent anode potential. Overall, this study helped understanding that the use of a non-capacitive electrode and intermittent anode potential deviated electrons to other processes other than electric current at the electrode by identifying electron sinks in the anolyte and quantifying the accumulation of electrons in the form of EPS.

Effective orthophosphate removal from surface water using hydrogen-oxidizing bacteria: Moving towards applicability.

Effective orthophosphate removal strategies are needed to counteract eutrophication and guarantee water quality. Previously, we established that hydrogen-oxidizing bacteria (HOB) have the ability to remove orthophosphate from artificial surface water. In the present study, we expand the application of the HOB orthophosphate removal strategy (1) to treat artificial surface water with low initial orthophosphate concentrations, (2) to treat real surface water and real wastewater effluent, and (3) to remove orthophosphate continuously. For synthetic surface water, irrespective of the initial concentration of 0.7, 0.5, 0.3, and 0.1 mg PO43--P/L, ultra-low concentrations (0.0058 ± 0.0028 mg PO43--P/L) were obtained. When artificial surface water was replaced by real surface water, without added nutrients or other chemicals, it was shown that over 90% orthophosphate could be removed within 30 min of operation in a batch configuration (0.031 ± 0.023 mg PO43--P/L). In continuous operation, orthophosphate removal from surface water left an average concentration of 0.040 ± 0.036 for 60 days, and the lowest orthophosphate concentration measured was 0.013 mg PO43-/L. Simultaneously, nitrate was continuously removed for 60 days below 0.1 mg/L. The ability to remove orthophosphate even under nitrogen limiting conditions might be related to the ability of HOB to fix nitrogen. This study brings valuable insights into the potential use of HOB biofilms for nutrient remediation and recovery.

Donnan Dialysis for scaling mitigation during electrochemical ammonium recovery from complex wastewater.

Inorganic scaling is often an obstacle for implementing electrodialysis systems in general and for nutrient recovery from wastewater specifically. In this work, Donnan dialysis was explored, to prevent scaling and to prolong operation of an electrochemical system for TAN (total ammonia nitrogen) recovery. An electrochemical system was operated with and without an additional Donnan dialysis cell, while being supplied with synthetic influent and real digested black water. For the same Load Ratio (nitrogen load vs applied current) while treating digested black water, the system operated for a period three times longer when combined with a Donnan cell. Furthermore, the amount of nitrogen recovered was higher. System performance was evaluated in terms of both TAN recovery and energy efficiency, at different Load Ratios. At a Load Ratio 1.3 and current density of 10 A m-2, a TAN recovery of 83% was achieved while consuming 9.7 kWh kgN-1.

Enrichment of Hydrogen Oxidizing Bacteria from High Temperature and Salinity Environments.

There is an urgent need for sustainable protein supply routes with low environmental footprint. Recently, the use of hydrogen oxidizing bacteria (HOB) as a platform for high quality microbial protein (MP) production has regained interest. This study aims to investigate the added value of using conditions such as salt and temperature to steer HOB communities to lower diversities, while maintaining a high protein content and a high quality amino acid profile. Pressure drop and hydrogen consumption were measured for 56 days to evaluate autotrophy of a total of six communities in serum flasks. Of the six communities, four were enriched under saline (0.0, 0.25, 0.5 and 1.0 mol NaCl l-1) and two under thermophilic conditions (65°C). Five communities enriched for HOB were subsequently cultivated in continuously stirred reactors under the same conditions to evaluate their potential as microbial protein producers. The protein percentages ranged from 41 to 80%. The highest protein content was obtained for the thermophilic enrichments. Amino acid profiles were comparable to protein sources commonly used for feed purposes. Members of the genus Achromobacter were found to dominate the saline enrichments while members of the genus Hydrogenibacillus were found to dominate the thermophilic enrichments. Here we show that enriching for HOB while steering the community toward low diversity and maintaining a high quality protein content can be successfully achieved, both in saline and thermophilic conditions.IMPORTANCE Alternative feed and food supply chains are required to decrease water and land use. HOB offer a promising substitute for traditional agricultural practice to produce microbial protein (MP) from residual materials and renewable energy. To safeguard product stability, the composition of the HOB community should be controlled. Defining strategies to maintain the stability of the communities is therefore key for optimization purposes. In this study, we use salt and temperature as independent conditions to stabilize the composition of the HOB communities. Based on the results presented, we conclude that HOB communities can be steered to have low diversity using the presented conditions while producing a desirable protein content with a valuable amino acid profile.

Minimal Bipolar Membrane Cell Configuration for Scaling Up Ammonium Recovery.

Electrochemical systems for total ammonium nitrogen (TAN) recovery are a promising alternative compared with conventional nitrogen-removal technologies. To make them competitive, we propose a new minimal stackable configuration using cell pairs with only bipolar membranes and cation-exchange membranes. The tested bipolar electrodialysis (BP-ED) stack included six cell pairs of feed and concentrate compartments. Critical operational parameters, such as current density and the ratio between applied current to nitrogen loading (load ratio), were varied to investigate the performance of the system using synthetic wastewater with a high nitrogen content as an influent (NH4 + ≈ 1.75 g L-1). High TAN removal (>70%) was achieved for a load ratio higher than 1. At current densities of 150 A m-2 and a load ratio of 1.2, a TAN transport rate of 1145.1±14.1 gN m-2 d-1 and a TAN-removal efficiency of 80% were observed. As the TAN removal was almost constant at different current densities, the BP-ED stack performed at a high TAN transport rate (819.1 gN m-2 d-1) while consuming the lowest energy (18.3 kJ gN -1) at a load ratio of 1.2 and 100 A m-2. The TAN transport rate, TAN removal, and energy input achieved by the minimal BP-ED stack demonstrated a promising new cell configuration for upscaling.

Enhanced Phototrophic Biomass Productivity through Supply of Hydrogen Gas.

Industrial production of phototrophic microorganisms is often hindered by low productivity due to limited light availability and therefore requires large land areas. This letter demonstrates that supply of hydrogen gas (H2) increases in phototrophic biomass productivity compared to a culture growing on light only. Experiments were performed growing Synechocystis sp. in batch bottles, with and without H2 in the headspace, which were exposed to light intensities of 70 and 100 µmol/m2/s. At 70 µmol/m2/s with H2, the average increase in biomass was 96 mg DW/L/d, whereas at 100 µmol/m2/s without H2, the average increase in biomass was 27 mg DW/L/d. Even at lower light intensity, the addition of H2 tripled the biomass yield compared to growth under light only. Photoreduction and photosynthesis occurred simultaneously, as both H2 consumption and O2 production were measured during biomass growth. Photoreduction used 1.85 mmol of H2 to produce 1.0 mmol of biomass, while photosynthesis produced 1.95 mmol of biomass. After transferring the culture to the dark, growth ceased, also in the presence of H2, showing that both light and H2 were needed for growth. A renewable H2 supply for higher biomass productivity is attractive since the combined efficiency of photovoltaics and electrolysis exceeds the photosynthetic efficiency.

Hydrogen oxidizing bacteria are capable of removing orthophosphate to ultra-low concentrations in a fed batch reactor configuration.

This paper proposes the use of hydrogen oxidizing bacteria (HOB) for the removal of orthophosphate from surface water as treatment step to prevent cyanobacterial blooms. To be effective as an orthophosphate removal strategy, an efficient transfer of hydrogen to the HOB is essential. A trickling filter was selected for this purpose. Using this system, a removal rate of 11.32 ± 0.43 mg PO4-3-P/L.d was achieved. The HOB biomass, developed on the trickling filter, is composed of 1.25% phosphorus on dry matter, which suggests that the orthophosphate removal principle is based on HOB growth. Cyanobacterial growth assays of the untreated and treated water showed that Synechocystis sp was only able to grow in the untreated water. Orthophosphate was removed to average residual values of 0.008 mg/L. In this proof of principle study, it is shown that HOB are able to remove orthophosphate from water to concentrations that prevent cyanobacterial growth.

Combination of bioelectrochemical systems and electrochemical capacitors: Principles, analysis and opportunities.

Bioelectrochemical systems combine electrodes and reactions driven by microorganisms for many different applications. The conversion of organic material in wastewater into electricity occurs in microbial fuel cells (MFCs). The power densities produced by MFCs are still too low for application. One way of increasing their performance is to combine them with electrochemical capacitors, widely used for charge storage purposes. Capacitive MFCs, i.e. the combination of capacitors and MFCs, allow for energy harvesting and storage and have shown to result in improved power densities, which facilitates the up scaling and application of the technology. This manuscript summarizes the state-of-the-art of combining capacitors with MFCs, starting with the theory and working principle of electrochemical capacitors. We address how different electrochemical measurements can be used to determine (bio)electrochemical capacitance and show how the measurement data can be interpreted. In addition, we present examples of the combination of electrochemical capacitors, both internal and external, that have been used to enhance MFC performance. Finally, we discuss the most promising applications and the main existing challenges for capacitive MFCs.

Mixed Culture Biocathodes for Production of Hydrogen, Methane, and Carboxylates.

Formation of hydrogen, methane, and organics at biocathodes is an attractive new application of bioelectrochemical systems (BESs). Using mixed cultures, these products can be formed at certain cathode potentials using specific operating conditions, of which pH is important. Thermodynamically, the reduction of CO2 to methane is the most favorable reaction, followed by reduction of CO2 to acetate and ethanol, and hydrogen. In practice, however, the cathode potential at which these reactions occur is more negative, meaning that more energy is required to drive the reaction. Therefore, hydrogen is often found as a second product or intermediate in the conversion of CO2 to both methane and carboxylates. In this chapter we summarize the inocula used for biocathode processes and discuss the achieved conversion rates and cathode potentials for formation of hydrogen, methane, and carboxylates. Although this overview reveals that BESs offer new opportunities for the bioproduction of different compounds, there are still challenges that need to be overcome before these systems can be applied on a larger scale. Graphical Abstract.

Haloalkaliphilic microorganisms assist sulfide removal in a microbial electrolysis cell.

Several industrial processes produce toxic sulfide containing streams that are often scrubbed using caustic solutions. An alternative, cost effective sulfide treatment method is bioelectrochemical sulfide removal. For the first time, a haloalkaliphilic sulfide-oxidizing microbial consortium was introduced to the anodic chamber of a microbial electrolysis cell operated at alkaline pH and with 1.0 M sodium ions. Under anode potential control, the highest sulfide removal rate was 2.16 mM/day and chemical analysis supported that the electrical current generation was from the sulfide oxidation. Biotic operation produced a maximum current density of 3625 mA/m2 compared to 210 mA/m2 while under abiotic operation. Furthermore, biotic electrical production was maintained for a longer period than for abiotic operation, potentially due to the passivation of the electrode by elemental sulfur during abiotic operation. The use of microorganisms reduced the energy input in this study compared to published electrochemical sulfide removal technologies. Sulfide-oxidizing populations dominated both the planktonic and electrode-attached communities with 16S rRNA gene sequences aligning within the genera Thioalkalivibrio, Thioalkalimicrobium, and Desulfurivibrio. The dominance of the Desulfurivibrio-like population on the anode surface offered evidence for the first haloalkaliphilic bacterium able to couple electrons from sulfide oxidation to extracellular electron transfer to the anode.

Microbial Community and Metabolic Activity in Thiocyanate Degrading Low Temperature Microbial Fuel Cells.

Thiocyanate is a toxic compound produced by the mining and metallurgy industries that needs to be remediated prior to its release into the environment. If the industry is situated at high altitudes or near the poles, economic factors require a low temperature treatment process. Microbial fuel cells are a developing technology that have the benefits of both removing such toxic compounds while recovering electrical energy. In this study, simultaneous thiocyanate degradation and electrical current generation was demonstrated and it was suggested that extracellular electron transfer to the anode occurred. Investigation of the microbial community by 16S rRNA metatranscriptome reads supported that the anode attached and planktonic anolyte consortia were dominated by a Thiobacillus-like population. Metatranscriptomic sequencing also suggested thiocyanate degradation primarily occurred via the 'cyanate' degradation pathway. The generated sulfide was metabolized via sulfite and ultimately to sulfate mediated by reverse dissimilatory sulfite reductase, APS reductase, and sulfate adenylyltransferase and the released electrons were potentially transferred to the anode via soluble electron shuttles. Finally, the ammonium from thiocyanate degradation was assimilated to glutamate as nitrogen source and carbon dioxide was fixed as carbon source. This study is one of the first to demonstrate a low temperature inorganic sulfur utilizing microbial fuel cell and the first to provide evidence for pathways of thiocyanate degradation coupled to electron transfer.

Energy-Efficient Ammonia Recovery in an Up-Scaled Hydrogen Gas Recycling Electrochemical System.

Nutrient and energy recovery is becoming more important for a sustainable future. Recently, we developed a hydrogen gas recycling electrochemical system (HRES) which combines a cation exchange membrane (CEM) and a gas-permeable hydrophobic membrane for ammonia recovery. This allowed for energy-efficient ammonia recovery, since hydrogen gas produced at the cathode was oxidized at the anode. Here, we successfully up-scaled and optimized this HRES for ammonia recovery. The electrode surface area was increased to 0.04 m2 to treat up to 11.5 L/day (∼46 gN/day) of synthetic urine. The system was operated stably for 108 days at current densities of 20, 50, and 100 A/m2. Compared to our previous prototype, this new cell design reduced the anode overpotential and ionic losses, while the use of an additional membrane reduced the ion transport losses. Overall, this reduced the required energy input from 56.3 kJ/gN (15.6 kW h/kgN) at 50 A/m2 (prototype) to 23.4 kJ/gN (6.5 kW h/kgN) at 100 A/m2 (this work). At 100 A/m2, an average recovery of 58% and a TAN (total ammonia nitrogen) removal rate of 598 gN/(m2 day) were obtained across the CEM. The TAN recovery was limited by TAN transport from the feed to concentrate compartment.