Ectoines production from biogas in pilot bubble column bioreactors and their subsequent extraction via bio-milking.

Despite the potential of biogas from waste/wastewater treatment as a renewable energy source, the presence of pollutants and the rapid decrease in the levelized cost of solar and wind power constrain the use of biogas for energy generation. Biogas conversion into ectoine, one of the most valuable bioproducts (1000 €/kg), constitutes a new strategy to promote a competitive biogas market. The potential for a stand-alone 20 L bubble column bioreactor operating at 6% NaCl and two 10 L interconnected bioreactors (at 0 and 6% NaCl, respectively) for ectoine production from biogas was comparatively assessed. The stand-alone reactor supported the best process performance due to its highest robustness and efficiency for ectoine accumulation (20-52 mgectoine/gVSS) and CH4 degradation (up to 84%). The increase in N availability and internal gas recirculation did not enhance ectoine synthesis. However, a 2-fold increase in the internal gas recirculation resulted in an approximately 1.3-fold increase in CH4 removal efficiency. Finally, the recovery of ectoine through bacterial bio-milking resulted in efficiencies of >70% without any negative impact of methanotrophic cell recycling to the bioreactors on CH4 biodegradation or ectoine synthesis.

Microbial Electrochemically Assisted Treatment Wetlands: Current Flow Density as a Performance Indicator in Real-Scale Systems in Mediterranean and Northern European Locations.

A METland is an innovative treatment wetland (TW) that relies on the stimulation of electroactive bacteria (EAB) to enhance the degradation of pollutants. The METland is designed in a short-circuit mode (in the absence of an external circuit) using an electroconductive bed capable of accepting electrons from the microbial metabolism of pollutants. Although METlands are proven to be highly efficient in removing organic pollutants, the study of in situ EAB activity in full-scale systems is a challenge due to the absence of a two-electrode configuration. For the first time, four independent full-scale METland systems were tested for the removal of organic pollutants and nutrients, establishing a correlation with the electroactive response generated by the presence of EAB. The removal efficiency of the systems was enhanced by plants and mixed oxic-anoxic conditions, with an average removal of 56 g of chemical oxygen demand (COD) mbed material -3 day-1 and 2 g of total nitrogen (TN) mbed material -3 day-1 for Ørby 2 (partially saturated system). The estimated electron current density (J) provides evidence of the presence of EAB and its relationship with the removal of organic matter. The tested METland systems reached the max. values of 188.14 mA m-2 (planted system; IMDEA 1), 223.84 mA m-2 (non-planted system; IMDEA 2), 125.96 mA m-2 (full saturated system; Ørby 1), and 123.01 mA m-2 (partially saturated system; Ørby 2). These electron flow values were remarkable for systems that were not designed for energy harvesting and unequivocally show how electrons circulate even in the absence of a two-electrode system. The relation between organic load rate (OLR) at the inlet and coulombic efficiency (CE; %) showed a decreasing trend, with values ranging from 8.8 to 53% (OLR from 2.0 to 16.4 g COD m-2 day-1) for IMDEA systems and from 0.8 to 2.5% (OLR from 41.9 to 45.6 g COD m-2 day-1) for Ørby systems. This pattern denotes that the treatment of complex mixtures such as real wastewater with high and variable OLR should not necessarily result in high CE values. METland technology was validated as an innovative and efficient solution for treating wastewater for decentralized locations.

Elucidating the key environmental parameters during the production of ectoines from biogas by mixed methanotrophic consortia.

Anaerobic digestion (AD) is a robust biotechnology for the valorisation of organic waste into biogas. However, the rapid decrease in renewable electricity prices requires alternative uses of biogas. In this context, the engineering of innovative platforms for the bio-production of chemicals from CH4 has recently emerged. The extremolyte and osmoprotectant ectoine, with a market price of ~1000€/Kg, is the industrial flagship of CH4-based bio-chemicals. This work aimed at optimizing the accumulation of ectoines using mixed microbial consortia enriched from saline environments (a salt lagoon and a salt river) and activated sludge, and biogas as feedstock. The influence of NaCl (0, 3, 6, 9 and 12 %) and Na2WO4 (0, 35 and 70 µg L-1) concentrations and incubation temperature (15, 25 and 35 °C) on the stoichiometry and kinetics of the methanotrophic consortia was investigated. Consortia enriched from activated sludge at 15 °C accumulated the highest yields of ectoine and hydroxyectoine at 6 % NaCl (105.0 ± 27.2 and 24.2 ± 5.4 mgextremolyte gbiomass-1, respectively). The consortia enriched from the salt lagoon accumulated the highest yield of ectoine and hydroxyectoine at 9 % NaCl (56.6 ± 2.5 and 51.0 ± 2.0 mgextremolyte gbiomass-1, respectively) at 25 °C. The supplementation of tungsten to the cultivation medium did not impact on the accumulation of ectoines in any of the consortia. A molecular characterization of the enrichments revealed a relative abundance of ectoine-accumulating methanotrophs of 7-16 %, with Methylomicrobium buryatense and Methylomicrobium japanense as the main players in the bioconversion of methane into ectoine.

Modeling and optimization strategies towards performance enhancement of microbial fuel cells.

Considering the complexity associated with bioelectrochemical processes, the performance of a microbial fuel cell (MFC) is governed by input operating parameters. For scaled-up applications, a MFC system needs to be modeled from engineering perspectives in terms of optimum operating conditions to get higher performance and energy recovery. Several conceptual numerical models to advanced computational simulation approaches have been developed to represent simple-form of a complex MFC system. Application of mathematical and computation models are explored to establish the relationship between operating input-variables and power output. The present review discusses about the complexity of system, modeling strategies used and reality of such modeling for scaling-up applications of MFCs. Additionally, the selection of an appropriate mathematical model reduces the computational duration and provides better understanding of the system process. It also explores the possibility and progress towards commercialization of MFCs and thus the need of development of model-based optimization and process-control approaches.

Innovative operational strategies in photosynthetic biogas upgrading in an outdoors pilot scale algal-bacterial photobioreactor.

Three innovative operational strategies were successfully evaluated to improve the quality of biomethane in an outdoors pilot scale photobioreactor interconnected to an external absorption unit: i) the use of a greenhouse during winter conditions, ii) a direct CO2 stripping in the photobioreactor via air stripping during winter conditions and iii) the use of digestate as make-up water during summer conditions. CO2 concentrations in the biomethane ranged from 0.4% to 6.1% using the greenhouse, from 0.3% to 2.6% when air was injected in the photobioreactor and from 0.4% to 0.9% using digestate as make up water. H2S was completely removed under all strategies tested. On the other hand, CH4 concentrations in biomethane ranged from 89.5% to 98.2%, from 93.0% to 98.2% and from 96.3% to 97.9%, when implementing strategies i), ii) and iii), respectively. The greenhouse was capable of maintaining microalgae productivities of 7.5 g m-2 d-1 during continental weather conditions, while mechanical CO2 stripping increased the pH in order to support an effective CO2 and H2S removal. Finally, the high evaporation rates during summer conditions allowed maintaining high inorganic carbon concentrations in the cultivation broth using centrate, which provided a cost-effective biogas upgrading.

Bidirectional microbial electron transfer: Switching an acetate oxidizing biofilm to nitrate reducing conditions.

Up to date a few electroactive bacteria embedded in biofilms are described to catalyze both anodic and cathodic reactions in bioelectrochemical systems (i.e. bidirectional electron transfer). How these bacteria transfer electrons to or from the electrode is still uncertain. In this study the extracellular electron transfer mechanism of bacteria within an electroactive biofilm was investigated by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). First, a mature anodic electroactive biofilm was developed from an activated sludge sample (inoculum), acetate as electron donor and a poised electrode (+397mV vs. SHE). Later, this biofilm was "switched" to biocathodic conditions by feeding it with a medium containing nitrates and poising the electrode at -303mV vs. SHE. The electrochemical characterization indicated that both, acetate oxidation and nitrate reduction took place at a similar formal potential of -175±05 and -175±34mV vs. SHE, respectively. The biofilm was predominantly composed by Geobacter sp. at both experimental conditions. Taken together, the results indicated that both processes could be catalyzed by using the same electron conduit, and most likely by the same bacterial consortium. Hence, this study suggests that electroactive bacteria within biofilms could use the same electron transfer conduit for catalyzing anodic and cathodic reactions.

Microbial characterization of anode-respiring bacteria within biofilms developed from cultures previously enriched in dissimilatory metal-reducing bacteria.

This work evaluated the use of a culture enriched in DMRB as a strategy to enrich ARB on anodes. DMRB were enriched with Fe(III) as final electron acceptor and then transferred to a potentiostatically-controlled system with an anode as sole final electron acceptor. Three successive iron-enrichment cultures were carried out. The first step of enrichment revealed a successful selection of the high current-producing ARB Geoalkalibacter subterraneus. After few successive enrichment steps, the microbial community analysis in electroactive biofilms showed a significant divergence with an impact on the biofilm electroactivity. Enrichment of ARB in electroactive biofilms through the pre-selection of DMRB should therefore be carefully considered.

Long-term continuous production of H2 in a microbial electrolysis cell (MEC) treating saline wastewater.

A biofilm-based 4 L two chamber microbial electrolysis cell (MEC) was continuously fed with acetate under saline conditions (35 g/L NaCl) for more than 100 days. The MEC produced a biogas highly enriched in H2 (≥90%). Both current (10.6 ± 0.2 A/m(2)Anode or 199.1 ± 4.0 A/m(3)MEC) and H2 production (201.1 ± 7.5 LH2/m(2)Cathode·d or 0.9 ± 0.0 m(3)H2/m(3)MEC·d) rates were highly significant when considering the saline operating conditions. A microbial analysis revealed an important enrichment in the anodic biofilm with five main bacterial groups: 44% Proteobacteria, 32% Bacteroidetes, 18% Firmicutes and 5% Spirochaetes and 1% Actinobacteria. Of special interest is the emergence within the Proteobacteria phylum of the recently described halophilic anode-respiring bacteria Geoalkalibacter (unk. species), with a relative abundance up to 14%. These results provide for the first time a noteworthy alternative for the treatment of saline effluents and continuous production of H2.

Specific and efficient electrochemical selection of Geoalkalibacter subterraneus and Desulfuromonas acetoxidans in high current-producing biofilms.

Two different saline sediments were used to inoculate potentiostatically controlled reactors (a type of microbial bioelectrochemical system, BES) operated in saline conditions (35 gNaCl l(-1)). Reactors were fed with acetate or a mixture of acetate and butyrate at two pH values: 7.0 or 5.5. Electroactive biofilm formation lag-phase, maximum current density production and coulombic efficiency were used to evaluate the overall performance of reactors. High current densities up to 8.5 A m(-2) were obtained using well-defined planar graphite electrodes. Additionally, biofilm microbial communities were characterized by CE-SSCP and 454 pyrosequencing. As a result of this procedure, two anode-respiring bacteria (ARB) always dominated the anodic biofilms: Geoalkalibacter subterraneus and/or Desulfuromonas acetoxidans. This suggests that a strong electrochemically driven selection process imposed by the applied potential occurs in the BES system. Moreover, the emergence of Glk. subterraneus in anodic biofilms significantly contributes to broaden the spectrum of high current producing microorganisms electrochemically isolated from environmental samples.

High current density via direct electron transfer by the halophilic anode respiring bacterium Geoalkalibacter subterraneus.

In this study the characterization of Geoalkalibacter subterraneus is presented, which is a novel halophilic anode respiring bacterium (ARB) previously selected and identified in a potentiostatically controlled bioelectrochemical system (BES) inoculated with sediments from a salt plant. Pure culture electroactive biofilms of Glk. subterraneus were grown during chronoamperometric batch experiments at a graphite electrode poised at +200 mV (vs. SCE) with 10 mM acetate as the electron donor. These biofilms exhibited the highest current density (4.68 ± 0.54 A m(-2)) reported on a planar material with a pure culture under saline conditions (3.5% NaCl). To investigate possible anodic electron transfer (ET) mechanisms, cyclic voltammetry (CV) of mature visible apparent reddish biofilms was performed under bioelectrocatalytic substrate consumption (turnover) and in the absence of the substrate (non-turnover). CV evidenced a well defined typical sigmoidal shape and a pair of clear redox couples under turnover and non-turnover conditions, respectively. Moreover, the calculation of their formal potentials indicated the presence of a common ET mechanism present in both CV conditions between -427.6 ± 0.5 (Ef,2) and -364.8 ± 4.5 mV (Ef,3). Confocal laser scanning microscopy inspection showed a biofilm structure composed of several layers of metabolically active bacteria that spread all over the electrode material within a biofilm up to 76 ± 7 µm thick. Such high value compared to the thickness values normally reported in the literature for pure culture electroactive bacteria justifies further investigations. Taken together, these results suggest that Glk. subterraneus performs a direct ET mechanism in contact with the electrode material. Furthermore, direct current generation from saline wastewater significantly expands the application of BESs.

Electron transfer and biofilm formation of Shewanella putrefaciens as function of anode potential.

Shewanellaceae are among the most widely studied electroactive microorganisms. In this report, we studied the influence of the applied electrode potential on the anodic current production of Shewanella putrefaciens NCTC 10695 under anoxic conditions. Furthermore, we used cyclic voltammetry (CV) and confocal laser scanning microscopy (CLSM) to investigate the microbial electron transfer and biofilm formation. It is shown that the chronoamperometric current density is increasing with increasing electrode potential from 3 µA cm(-2) at -0.1 V up to -12 µA cm(-2) at +0.4 V (vs. Ag/AgCl), which is accompanied by an increasing amount of biomass deposited on the electrode. By means of cyclic voltammetry we demonstrate that direct electron transfer (DET) is dominating and the planktonic cells play only a minor role.

Electroactive mixed culture derived biofilms in microbial bioelectrochemical systems: the role of pH on biofilm formation, performance and composition.

The pH-value played a crucial role for the development and current production of anodic microbial electroactive biofilms. It was demonstrated that only a narrow pH-window, ranging from pH 6 to 9, was suitable for growth and operation of biofilms derived from pH-neutral wastewater. Any stronger deviation from pH neutral conditions led to a substantial decrease in the biofilm performance. Thus, average current densities of 151, 821 and 730 µA cm(-2) were measured for anode biofilms grown and operated at pH 6, 7 and 9 respectively. The microbial diversity of the anode chamber community during the biofilm selection process was studied using the low cost method flow-cytometry. Thereby, it was demonstrated that the pH value as well as the microbial inocula had an impact on the resulting anode community structure. As shown by cyclic voltammetry the electron transfer thermodynamics of the biofilms was strongly depending on the solution's pH-value.

Cyclic voltammetric analysis of the electron transfer of Shewanella oneidensis MR-1 and nanofilament and cytochrome knock-out mutants.

Shewanella is frequently used as a model microorganism for microbial bioelectrochemical systems. In this study, we used cyclic voltammetry (CV) to investigate extracellular electron transfer mechanisms from S. oneidensis MR-1 (WT) and five deletion mutants: membrane bound cytochrome (∆mtrC/ΔomcA), transmembrane pili (ΔpilM-Q, ΔmshH-Q, and ΔpilM-Q/ΔmshH-Q) and flagella (∆flg). We demonstrate that the formal potentials of mediated and direct electron transfer sites of the derived biofilms can be gained from CVs of the respective biofilms recorded at bioelectrocatlytic (i.e. turnover) and lactate depleted (i.e. non-turnover) conditions. As the biofilms possess only a limited bioelectrocatalytic activity, an advanced data processing procedure, using the open-source software SOAS, was applied. The obtained results indicate that S. oneidensis mutants used in this study are able to bypass hindered direct electron transfer by alternative redox proteins as well as self-mediated pathways.