Enriching electroactive microorganisms from ferruginous lake waters - Mind the sulfate reducers!

Electroactive microorganisms are pivotal players in mineral transformation within redox interfaces characterized by pronounced oxygen and dissolved metal gradients. Yet, their systematic cultivation from such environments remains elusive. Here, we conducted an anodic enrichment using anoxic ferruginous waters from a post-mining lake as inoculum. Weak electrogenicity (j = ∼5 µA cm-2) depended on electroactive planktonic cells rather than anodic biofilms, with a preference for formate as electron donor. Addition of yeast extract decreased the lag phase but did not increase current densities. The enriched bacterial community varied depending on the substrate composition but mainly comprised of sulfate- and nitrate-reducing bacteria (e.g., Desulfatomaculum spp. and Stenotrophomonas spp.). A secondary enrichment strategy resulted in different bacterial communities composed of iron-reducing (e.g., Klebsiella spp.) and fermentative bacteria (e.g., Paeniclostridium spp.). Secondary electron microscopy and energy-dispersive X-ray spectroscopy results indicate the precipitation of sulfur- and iron-rich organomineral aggregates at the anode surface, presumably impeding current production. Our findings indicate that (i) anoxic waters containing geogenically derived metals can be used to enrich weak electricigens, and (ii) it is necessary to specifically inhibit sulfate reducers. Otherwise, sulfate reducers tend to dominate over EAM during cultivation, which can lead to anode passivation due to biomineralization.

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.

Improving the performance of bioelectrochemical sulfate removal by applying flow mode.

Treatment of wastewater contaminated with high sulfate concentrations is an environmental imperative lacking a sustainable and environmental friendly technological solution. Microbial electrochemical technology (MET) represents a promising approach for sulfate reduction. In MET, a cathode is introduced as inexhaustible electron source for promoting sulfate reduction via direct or mediated electron transfer. So far, this is mainly studied in batch mode representing straightforward and easy-to-use systems, but their practical implementation seems unlikely, as treatment capacities are limited. Here, we investigated bioelectrochemical sulfate reduction in flow mode and achieved removal efficiencies (Esulfate , 89.2 ± 0.4%) being comparable to batch experiments, while sulfate removal rates (Rsulfate , 3.1 ± 0.2 mmol L-1 ) and Coulombic efficiencies (CE, 85.2 ± 17.7%) were significantly increased. Different temperatures and hydraulic retention times (HRT) were applied and the best performance was achieved at HRT 3.5 days and 30°C. Microbial community analysis based on amplicon sequencing demonstrated that sulfate reduction was mainly performed by prokaryotes belonging to the genera Desulfomicrobium, Desulfovibrio, and Desulfococcus, indicating that hydrogenotrophic and heterotrophic sulfate reduction occurred by utilizing cathodically produced H2 or acetate produced by homoacetogens (Acetobacterium). The advantage of flow operation for bioelectrochemical sulfate reduction is likely based on higher absolute biomass, stable pH, and selection of sulfate reducers with a higher sulfide tolerance, and improved ratio between sulfate-reducing prokaryotes and homoacetogens.

Electrochemical and Microbial Dissection of Electrified Biotrickling Filters.

Electrified biotrickling filters represent sustainable microbial electrochemical technology for treating organic carbon-deficient ammonium-contaminated waters. However, information on the microbiome of the conductive granule bed cathode remains inexistent. For uncovering this black box and for identifying key process parameters, minimally invasive sampling units were introduced, allowing for the extraction of granules from different reactor layers during reactor operation. Sampled granules were analyzed using cyclic voltammetry and molecular biological tools. Two main redox sites [-288 ± 18 mV and -206 ± 21 mV vs. standard hydrogen electrode (SHE)] related to bioelectrochemical denitrification were identified, exhibiting high activity in a broad pH range (pH 6-10). A genome-centric analysis revealed a complex nitrogen food web and the presence of typical denitrifiers like Pseudomonas nitroreducens and Paracoccus versutus with none of these species being identified as electroactive microorganism so far. These are the first results to provide insights into microbial structure-function relationships within electrified biotrickling filters and underline the robustness and application potential of bioelectrochemical denitrification for environmental remediation.

Redox Potential Heterogeneity in Fixed-Bed Electrodes Leads to Microbial Stratification and Inhomogeneous Performance.

Bed electrodes provide high electrode area-to-volume ratios represent a promising configuration for transferring bioelectrochemical systems close to industrial applications. Nevertheless, the intrinsic electrical resistance leads to poor polarization behavior. Therefore, the distribution of Geobacter spp. and their electrochemical performance within exemplary fixed-bed electrodes are investigated. A minimally invasive sampling system allows characterization of granules from different spatial locations of bed electrodes. Cyclic voltammetry of single granules (n=63) demonstrates that the major share of electroactivity (134.3 mA L-1 ) is achieved by approximately 10 % of the bed volume, specifically that being close to the current collector. Nevertheless, analysis of the microbial community reveals that Geobacter spp. dominated all sampled granules. These findings clearly demonstrate the need for engineered bed electrodes to improve electron exchange between microorganisms and granules.

Electro-bioremediation of nitrate and arsenite polluted groundwater.

The coexistence of different pollutants in groundwater is a common threat. Sustainable and resilient technologies are required for their treatment. The present study aims to evaluate microbial electrochemical technologies (METs) for treating groundwater contaminated with nitrate (NO3-) while containing arsenic (in form of arsenite (As(III)) as a co-contaminant. The treatment was based on the combination of nitrate reduction to dinitrogen gas and arsenite oxidation to arsenate (exhibiting less toxicity, solubility, and mobility), which can be removed more easily in further post-treatment. We operated a bioelectrochemical reactor at continuous-flow mode with synthetic contaminated groundwater (33 mg N-NO3- L-1 and 5 mg As(III) L-1) identifying the key operational conditions. Different hydraulic retention times (HRT) were evaluated, reaching a maximum nitrate reduction rate of 519 g N-NO3- m3Net Cathodic Compartment d-1 at HRT of 2.3 h with a cathodic coulombic efficiency of around 100 %. Simultaneously, arsenic oxidation was complete at all HRT tested down to 1.6 h reaching an oxidation rate of up to 90 g As(III) m-3Net Reactor Volume d -1. Electrochemical and microbiological characterization of single granules suggested that arsenite at 5 mg L-1 did not have an inhibitory effect on a denitrifying biocathode mainly represented by Sideroxydans sp. Although the coexistence of abiotic and biotic arsenic oxidation pathways was shown to be likely, microbial arsenite oxidation linked to denitrification by Achromobacter sp. was the most probable pathway. This research paves the ground towards a real application for treating groundwater with widespread pollutants.

Electrifying biotrickling filters for the treatment of aquaponics wastewater.

This work aimed to study the electrification of biotrickling filters by means of Microbial electrochemical technologies (MET) to develop an easy-to-assemble and easy-to-use MET for nitrogen removal without external aeration nor addition of chemicals. Four different designs were tested. The highest ammonium and nitrate removal rates (94 gN·m-3·d-1 and 43 gN·m-3·d-1, respectively) were reached by combining an aerobic zone with an electrified anoxic zone. The standards of effluent quality suitable for hydroponics were met at low energy cost (8.3 × 10-2 kWh·gN-1). Electrified biotrickling filters are a promising alternative for aquaponics and a potential treatment for organic carbon-deficient ammonium-contaminated waters.

Determining incremental coulombic efficiency and physiological parameters of early stage Geobacter spp. enrichment biofilms.

Geobacter spp. enrichment biofilms were cultivated in batch using one-chamber and two-chamber bioelectrochemical reactors. Time-resolved substrate quantification was performed to derive physiological parameters as well as incremental coulombic efficiency (i.e., coulombic efficiency during one batch cycle, here every 6h) during early stage biofilm development. The results of one-chamber reactors revealed an intermediate acetate increase putatively due to the presence of acetogens. Total coulombic efficiencies of two-chamber reactors were considerable lower (19.6±8.3% and 49.3±13.2% for 1st and 2nd batch cycle, respectively) compared to usually reported values of mature Geobacter spp. enrichment biofilms presumably reflecting energetic requirements for biomass production (i.e., cells and extracellular polymeric substances) during early stages of biofilm development. The incremental coulombic efficiency exhibits considerable changes during batch cycles indicating shifts between phases of maximizing metabolic rates and maximizing biomass yield. Analysis based on Michaelis-Menten kinetics yielded maximum substrate uptake rates (vmax,Ac, vmax,I) and half-saturation concentration coefficients (KM,Ac,KM,I) based on acetate uptake or current production, respectively. The latter is usually reported in literature but neglects energy demands for biofilm growth and maintenance as well as acetate and electron storage. From 1st to 2nd batch cycle, vmax,Ac and KM,Ac, decreased from 0.0042-0.0051 mmol Ac- h-1 cm-2 to 0.0031-0.0037 mmol Ac- h-1 cm-2 and 1.02-2.61 mM Ac- to 0.28-0.42 mM Ac-, respectively. Furthermore, differences between KM,Ac/KM,I and vmax,Ac/vmax,I were observed providing insights into the physiology of Geobacter spp. enrichment biofilms. Notably, KM,I considerably scattered while vmax,Ac/vmax,I and KM,Ac remained rather stable indicating that acetate transport within biofilm only marginally affects reaction rates. The observed data variation mandates the requirement of a more detailed analysis with an improved experimental system, e.g., using flow conditions and a comparison with Geobacter spp. pure cultures.

Corrigendum: Spotlight on the Energy Harvest of Electroactive Microorganisms: The Impact of the Applied Anode Potential.

[This corrects the article DOI: 10.3389/fmicb.2019.01352.].

Spotlight on the Energy Harvest of Electroactive Microorganisms: The Impact of the Applied Anode Potential.

Electroactive microorganisms (EAM) harvest energy by reducing insoluble terminal electron acceptors (TEA) including electrodes via extracellular electron transfer (EET). Therefore, compared to microorganisms respiring soluble TEA, an adapted approach is required for thermodynamic analyses. In EAM, the thermodynamic frame (i.e., maximum available energy) is restricted as only a share of the energy difference between electron donor and TEA is exploited via the electron-transport chain to generate proton-motive force being subsequently utilized for ATP synthesis. However, according to a common misconception, the anode potential is suggested to co-determine the thermodynamic frame of EAM. By comparing the model organism Geobacter spp. and microorganisms respiring soluble TEA, we reason that a considerable part of the electron-transport chain of EAM performing direct EET does not contribute to the build-up of proton-motive force and thus, the anode potential does not co-determine the thermodynamic frame. Furthermore, using a modeling platform demonstrates that the influence of anode potential on energy harvest is solely a kinetic effect. When facing low anode potentials, NADH is accumulating due to a slow direct EET rate leading to a restricted exploitation of the thermodynamic frame. For anode potentials ≥ 0.2 V (vs. SHE), EET kinetics, NAD+/NADH ratio as well as exploitation of the thermodynamic frame are maximized, and a further potential increase does not result in higher energy harvest. Considering the limited influence of the anode potential on energy harvest of EAM is a prerequisite to improve thermodynamic analyses, microbial resource mining, and to transfer microbial electrochemical technologies (MET) into practice.

Modeling Microbial Electrosynthesis.

Mathematical modeling is an overarching approach for assessing the complexity of microbial electrosynthesis (MES) and for complementing the relevant experimental research. By describing and linking compartments, components, and processes with appropriate mathematical equations, MES and the corresponding bioelectrodes and complete bioelectrochemical systems can be analyzed and predicted across several temporal and local scales. Thereby, insights into fundamental phenomena and mechanisms, in addition to process engineering and design can be obtained. However, a substantial lack of knowledge about extracellular electron transfer mechanisms and electrotrophic microorganisms presumably prevented the development of adequate models of MES, especially of biocathodes, so far. To propel efforts regarding this demanding task, this chapter provides a comprehensive overview of the relevant compartments, components and processes, appropriate model strategies, and a discussion on potential modeling pitfalls. By adapting an established approach to assessing the energetics of microorganism, an instruction for calculating stoichiometry, thermodynamics, and kinetics, with the example of electro-autotrophic growth at cathodes, is presented. Models of bioanodes and fundamental electrochemical equations are described to provided strategies for calculating cathodic electron-uptake reactions and connecting them to the microbial metabolism. Finally, differential equations are detailed for coupling the distinct compartments of a bioelectrochemical system. Although MES comprises anodic and cathodic reactions, the present chapter focuses on biocathodes representing a functional connection between cathode and electron-accepting microorganisms. Graphical Abstract.

Microbial ecology-based engineering of Microbial Electrochemical Technologies.

Microbial ecology is devoted to the understanding of dynamics, activity and interaction of microorganisms in natural and technical ecosystems. Bioelectrochemical systems represent important technical ecosystems, where microbial ecology is of highest importance for their function. However, whereas aspects of, for example, materials and reactor engineering are commonly perceived as highly relevant, the study and engineering of microbial ecology are significantly underrepresented in bioelectrochemical systems. This shortfall may be assigned to a deficit on knowledge and power of these methods as well as the prerequisites for their thorough application. This article discusses not only the importance of microbial ecology for microbial electrochemical technologies but also shows which information can be derived for a knowledge-driven engineering. Instead of providing a comprehensive list of techniques from which it is hard to judge the applicability and value of information for a respective one, this review illustrates the suitability of selected techniques on a case study. Thereby, best practice for different research questions is provided and a set of key questions for experimental design, data acquisition and analysis is suggested.

A framework for modeling electroactive microbial biofilms performing direct electron transfer.

A modeling platform for microbial electrodes based on electroactive microbial biofilms performing direct electron transfer (DET) is presented. Microbial catabolism and anabolism were coupled with intracellular and extracellular electron transfer, leading to biofilm growth and current generation. The model includes homogeneous electron transfer from cells to a conductive biofilm component, biofilm matrix conduction, and heterogeneous electron transfer to the electrode. Model results for Geobacter based anodes, both at constant electrode potential and in voltammetric (dynamic electrode potential) conditions, were compared to experimental data from different sources. The model can satisfactorily describe microscale (concentration, pH and redox gradients) and macroscale (electric currents, biofilm thickness) properties of Geobacter biofilms. The concentration of electrochemically accessible redox centers, here denominated as cytochromes, involved in the extracellular electron transfer, plays the key role and may differ between constant potential (300 mM) and dynamic potential (3mM) conditions. Model results also indicate that the homogeneous and heterogeneous electron transfer rates have to be within the same order of magnitude (1.2 s(-1)) for reversible extracellular electron transfer.