Revealing cellular (poly)sulphide storage in electrochemically active sulphide oxidising bacteria using rotating disc electrodes.

Sulphide oxidising bacteria (SOB) have the potential to be used for bioelectrochemical removal, i.e. oxidation, of sulphide from waste streams. In anaerobic conditions, SOB are able to spatially separate sulphide removal and terminal electron transfer to an electrode and act as a sulphide shuttle. However, it is not fully understood how SOB anaerobically remove sulphide and store charge equivalents, and where in this process sulphur is formed. Therefore, the redox behaviour of sulphide shuttling SOB was investigated at haloalkaline conditions using a glassy carbon rotating disc electrode (RDE) and cyclic voltammetry. Voltammograms of SOB in the absence and presence of sulphide were compared to voltammograms of abiotic sulphur species solutions. Polysulphide and sulphide showed different redox behaviour, with distinct potentials for oxidation of > -0.3 V (vs. Ag/AgCl) for polysulphide and > -0.1 V for sulphide. Comparing biotic to abiotic experiments lead to the hypothesis that SOB formed polysulphides during anaerobic sulphide removal, which stayed sorbed to the cells. With this study, further steps were taken in elucidating the mechanisms of sulphide shuttling by SOB.

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.

Enhancement of Ammonium Oxidation at Microoxic Bioanodes.

Bioelectrochemical systems (BESs) are considered to be energy-efficient to convert ammonium, which is present in wastewater. The application of BESs as a technology to treat wastewater on an industrial scale is hindered by the slow removal rate and lack of understanding of the underlying ammonium conversion pathways. This study shows ammonium oxidation rates up to 228 ± 0.4 g-N m-3 d-1 under microoxic conditions (dissolved oxygen at 0.02-0.2 mg-O2/L), which is a significant improvement compared to anoxic conditions (120 ± 21 g-N m-3 d-1). We found that this enhancement was related to the formation of hydroxylamine (NH2OH), which is rate limiting in ammonium oxidation by ammonia-oxidizing microorganisms. NH2OH was intermediate in both the absence and presence of oxygen. The dominant end-product of ammonium oxidation was dinitrogen gas, with about 75% conversion efficiency in the presence of a microoxic level of dissolved oxygen and 100% conversion efficiency in the absence of oxygen. This work elucidates the dominant pathways under microoxic and anoxic conditions which is a step toward the application of BESs for ammonium removal in wastewater treatment.

Effect of organic amendments obtained from different pretreatment technologies on soil microbial community.

The application of organic amendments (OAs) obtained from biological treatment technologies is a common agricultural practice to increase soil functionality and fertility. OAs and their respective pretreatment processes have been extensively studied. However, comparing the properties of OAs obtained from different pretreatment processes remains challenging. In most cases, the organic residues used to produce OAs exhibit intrinsic variability and differ in origin and composition. In addition, few studies have focused on comparing OAs from different pretreatment processes in the soil microbiome, and the extent to which OAs affect the soil microbial community remains unclear. This limits the design and implementation of effective pretreatments aimed at reusing organic residues and facilitating sustainable agricultural practices. In this study, we used the same model residues to produce OAs to enable meaningful comparisons among compost, digestate, and ferment. These three OAs contained different microbial communities. Compost had higher bacterial but lower fungal alpha diversity than ferment and digestate. Compost-associated microbes were more prevalent in the soil than ferment- and digestate-associated microbes. More than 80% of the bacterial ASVs and fungal OTUs from the compost were detected 3 months after incorporation into the soil. However, the addition of compost had less influence on the resulting soil microbial biomass and community composition than the addition of ferment or digestate. Specific native soil microbes, members from Chloroflexi, Acidobacteria, and Mortierellomycota, were absent after ferment and digestate application. The addition of OAs increased the soil pH, particularly in the compost-amended soil, whereas the addition of digestate enhanced the concentrations of dissolved organic carbon (DOC) and available nutrients (such as ammonium and potassium). These physicochemical variables were key factors that influenced soil microbial communities. This study furthers our understanding of the effective recycling of organic resources for the development of sustainable soils.

Nutrient recovery and pollutant removal during renewable fuel production: opportunities and challenges.

Stimulated by the desire to achieve a Net Zero energy economy, the demand for renewable fuels is growing rapidly. The production of toxic waste streams that accompanies the transition from fossil fuels to renewable fuels is often overlooked. These waste streams include, among others, thiols and ammonia, and benzene, toluene, and xylene (BTX). When suitable treatment technologies are available, these compounds can be converted to valuable nutrients. In this opinion article, we provide an overview of expected waste streams and their characteristics. We indicate future challenges for associated waste streams, such as the lag in developing resource recovery technologies. Furthermore, we discuss unexploited opportunities to recover valuable nutrients from these waste streams.

Anaerobic sulphide removal by haloalkaline sulphide oxidising bacteria.

Sulphide is a toxic and corrosive compound and requires removal from waste streams. Recent discoveries show that sulphide oxidising bacteria (SOB) from modern desulphurisation plants are able to spatially separate sulphide removal and oxygen reduction when exposed to intermittent anaerobic and aerobic environments. Here, SOB act as electron shuttles between electron donor and acceptor. The underlying mechanisms for electron shuttling are of yet unknown. To investigate the anaerobic sulphide removal of SOB, batch experiments and mathematical models were applied. The sulphide removal capacity decreased at increasing biomass concentrations. At 0.6 mgN/L SOB could remove up to 8 mgS/mgN in 30 min. It was found that biological activity determines sulphide removal, alongside chemical processes. Anaerobic oxidation of electron carriers was determined to only explain 0.1% of charge storage, where irreversible cleavage of long chain polysulphides could explain full sulphide storage. Different sulphide removal and intracellular storage processes are postulated.

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.

The effect of anode potential on electrogenesis, methanogenesis and sulfidogenesis in a simulated sewer condition.

Methane emissions from the sewer system are considered to be a non-negligible source of aggravating the greenhouse effect. Meanwhile, the sewer system has long been plagued by sulfide-induced corrosion problems. This study explored the possibility of using a bioelectrochemical system to intensify the competition between electroactive bacteria, methanogens and sulfate-reducing bacteria, thereby reducing the production of methane and sulfide. Dual-chamber bioelectrochemical reactors were constructed and operated in fed-batch mode with the coexistence of Electroactive bacteria, Methanogenic archaea and Sulfate-reducing bacteria. Acetate was supplied as the sole carbon source. The results indicated that electrogenesis induced by the anode potentials of -0.42 V and -0.2 V (vs. Ag/AgCl) had advantages over methanogenesis and sulfidogenesis in consuming acetate. The stimulated electrogenesis by anode potentials resulted in a decrease in pH. Methane production was suppressed in the reactors with anode potentials of -0.42 and -0.2 V compared to open circuit controls. In contrast to methane, the capacity for sulfide production was facilitated in the reactors with the anode potentials of -0.42 V and -0.2 V compared to open circuit controls. 16s rRNA gene analysis showed that Geobacter was the most abundant genus on the anode biofilm in the anode potential-controlled reactor, while acetoclastic methanogens dominated in open circuit controls. Methanosaeta and Methanosarcina were the most abundant methanogens in open circuit controls. Collectively, our study demonstrates that the use of electrodes with anode potential control can help to control methane emissions, but could not yet prevent sulfide production, which requires further research.

A simple method for routine measurement of organosulfur compounds in complex liquid and gaseous matrices.

The measurement of VOSCs in complex matrices is challenging due to their volatile and reactive nature. A straightforward method using headspace chromatography was developed for routine analyses of organosulfur compounds in a high saline liquid matrix with a pH of 8.4. Direct sample acidification with a 1M acetate buffer (pH 3.6) showed an increased response for methanethiol, ethanethiol, propanethiol, dimethyl sulfide, dimethyl disulfide and diethyl disulfide. A good quadratic fit (R2 <0.995) was obtained for each compound over a calibration range of 5 µM-S until 125 µM-S (µmol sulfur/L). Gas standards were measured using the same chromatographic conditions over a calibration range of 0.08 µM-S until 1.85 µM-S (R2 <0.999). Gas standards could also be used to calibrate the liquid phase with a response ratio of 105.2% for ET, 107% for DMS, 105.7% for PT, 108.9% for DMDS and 106% for DEDS. This alternative calibration strategy reduced the preparation time and does not rely on liquid standards, which were unstable over time. This method was used to determine Henry constants for the organosulfur compounds both in demineralized water and the high saline liquid matrix and to analyze samples from a bio electrochemical experiment that treated methanethiol. This new method allows for routine analysis of samples originating from natural gas desulfurization plants and can potentially also be used to analyze organosulfur compounds in other complex waste streams.

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.

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.

Reduced overpotential of methane-producing biocathodes: Effect of current and electrode storage capacity.

Cathode overpotential is a key factor in the energy efficiency of bioelectrochemical systems. In this study the aim is to demonstrate the role of applied current density and electrode storage capacity on cathode overpotential. To do so, eight reactors using capacitive granular activated carbon as cathode material were operated. Four reactors were controlled at -5 A m-2 and four at -10 A m-2. Additionally, to evaluate the electrode storage capacity, weekly charge/discharge tests were conducted for half of the reactors at each applied current density. Results show that cathode potential as high as -0.50 V vs. Ag/AgCl can be reached. Furthermore, the resulting low cathode overpotential is both dependent on applied current density and employment (or not) of charge/discharge tests: reactors at -10 A m-2 without charge/discharge regimes did not result in increasing cathode potential whereas reactors at -5 A m-2 and at -10 A m-2 with charge/discharge regimes did.

Continuous electron shuttling by sulfide oxidizing bacteria as a novel strategy to produce electric current.

Sulfide oxidizing bacteria (SOB) are widely applied in industry to convert toxic H2S into elemental sulfur. Haloalkaliphilic planktonic SOB can remove sulfide from solution under anaerobic conditions (SOB are 'charged'), and release electrons at an electrode (discharge of SOB). The effect of this electron shuttling on product formation and biomass growth is not known. Here, we study and demonstrate a continuous process in which SOB remove sulfide from solution in an anaerobic 'uptake chamber', and shuttle these electrons to the anode of an electrochemical cell, in the absence of dissolved sulfide. Two experiments over 31 and 41 days were performed. At a sulfide loading rate of 1.1 mmolS/day, electricity was produced continuously (3 A/m2) without dissolved sulfide in the anolyte. The main end product was sulfate (56% in experiment 1% and 78% in experiment 2), and 87% and 77% of the electrons in sulfide were recovered as electricity. It was found that the current density was dependent on the sulfide loading rate and not on the anode potential. Biological growth occurred, mainly at the anode as biofilm, in which the deltaproteobacterial genus Desulfurivibrio was dominating. Our results demonstrate a novel strategy to produce electricity from sulfide in an electrochemical system.

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.

Effect of nitrogen, phosphorus and pH on biological wood oxidation at 42 °C.

Biological wood oxidation (BWO) is proposed as a cleaner alternative to wood combustion for heat production and wood waste management. Currently, BWO is not extensively studied and little is known about it. Nevertheless, given the composition of wood residues, which is dominated by carbon, nutrient availability may become a limiting factor during BWO. Our objective was to study the nutrition requirements for sustaining the BWO. For this purpose, three different factors including nitrogen addition, phosphorus addition and pH, were studied. Oxygen consumption and mass loss were monitored and used to evaluate the impact of nutrition on BWO and to calculate the theoretical heat production. The result showed that nitrogen addition at a relatively low level (2.5-10 mg/g) enhanced the cumulative oxygen consumption by 60-124% and mass loss by 28-95%, when compared with the BWO without nitrogen addition. The highest nitrogen addition examined in this research (20 mg/g), on the other hand, did not enhance BWO. Different phosphorus addition (0.5-5 mg/g) and pH (4-6) had little impacts on BWO. The highest theoretical heat production rate (0.63 W/kg dry wood biomass) was achieved using 2.5 mg/g nitrogen addition with a 95-day incubation. This suggests that nitrogen addition is required and able to sustain BWO. Besides, the cumulative oxygen consumption showed a good linear relationship with mass loss. This study provides the first indication on the effective quantify of nitrogen addition for enhancing BWO, which contributes to the selection of nutrient source for BWO in future studies.

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.

Biologically enhanced hydrogen sulfide absorption from sour gas under haloalkaline conditions.

We studied a biotechnological desulfurization process for removal of toxic hydrogen sulfide (H2S) from sour gas. The process consists of two steps: i) Selective absorption of H2S into a (bi)carbonate solution in the absorber column and ii) conversion of sulfide to sulfur by sulfide oxidizing bacteria (SOB) in the aerated bioreactor. In previous studies, several physico-chemical factors were assessed to explain the observed enhancement of H2S absorption in the absorber, but a full explanation was not provided. We investigated the relation between the metabolic activity of SOB and the enhancement factor. Two continuous experiments on pilot-scale were performed to determine H2S absorption efficiencies at different temperatures and biomass concentrations. The absorption efficiency improved at increasing temperatures, i.e. H2S concentration in the treated gas decreased from 715 ±â€¯265 ppmv at 25.4 °C to 69 ±â€¯25 ppmv at 39.4 °C. The opposite trend is expected when H2S absorption is solely determined by physico-chemical factors. Furthermore, increasing biomass concentrations to the absorber also resulted in decreased H2S concentrations in the treated gas, from approximately 6000 ppmv without biomass to 1664 ±â€¯126 ppmv at 44 mg N/L. From our studies it can be concluded that SOB activity enhances H2S absorption and leads to increased H2S removal efficiencies in biotechnological gas desulfurization.

3D biofilm visualization and quantification on granular bioanodes with magnetic resonance imaging.

The use of microbial fuel cells (MFCs) for wastewater treatment fits in a circular economy context, as they can produce electricity by the removal of organic matter in the wastewater. Activated carbon (AC) granules are an attractive electrode material for bioanodes in MFCs, as they are cheap and provide electroactive bacteria with a large surface area for attachment. The characterization of biofilm growth on AC granules, however, is challenging due to their high roughness and three-dimensional structure. In this research, we show that 3D magnetic resonance imaging (MRI) can be used to visualize biofilm distribution and determine its volume on irregular-shaped single AC granules in a non-destructive way, while being combined with electrochemical and biomass analyses. Ten AC granules with electroactive biofilm (i.e. granular bioanodes) were collected at different growth stages (3 to 21 days after microbial inoculation) from a multi-anode MFC and T1-weighted 3D-MRI experiments were performed for three-dimensional biofilm visualization. With time, a more homogeneous biofilm distribution and an increased biofilm thickness could be observed in the 3D-MRI images. Biofilm volumes varied from 0.4 µL (day 4) to 2 µL (day 21) and were linearly correlated (R2 = 0.9) to the total produced electric charge and total nitrogen content of the granular bioanodes, with values of 66.4 C µL-1 and 17 µg N µL-1, respectively. In future, in situ MRI measurements could be used to monitor biofilm growth and distribution on AC granules.