Flow-through laminar anodes with variable interlaminar distance to modulate the current density of urine-fed bio-electrochemical systems.

Three-dimensional (3D) porous anodes used in urine-powered bio-electrochemical applications usually lead to the growth of electro-active bacteria on the outer electrode surface, due to limited microbial access to the internal structure and lack of permeation of culture medium through the entire porous architecture. In this study, we propose the use of 3D monolithic Ti4O7 porous electrodes with controlled laminar structures as microbial anodes for urine-fed bio-electrochemical systems. The interlaminar distance was tuned to modulate the anode surface areas and, thus, the volumetric current densities. To profit from the true area of the electrodes, urine feeding was performed as a continuous flow through the laminar architectures. The system was optimized according to the response surface methodology (RSM). The electrode interlaminar distance and the concentration of urine were selected as independent variables, with the volumetric current density as the output response to optimize. Maximum current densities of 5.2 kA.m-3 were produced from electrodes with 12 µm-interlaminar distance and 10 %v/v urine concentrations. The present study demonstrates the existence of a trade-off between the accesibility to the internal electrode structure and the effective usage of the surface area to maximize the volumetric current density when diluted urine is used as flowing-through feeding fuel.

Energetics, electron uptake mechanisms and limitations of electroautotrophs growing on biocathodes - A review.

Electroautotrophs are microorganisms that can take the electrons needed for energy generation, CO2 fixation and other metabolic reactions from a polarized electrode. They have been the focus of intense research for its application in wastewater treatment, bioelectrosynthetic processes and hydrogen generation. As a general trend, current densities produced by the electron uptake of these microorganisms are low, limiting their applicability at large scale. In this work, the electron uptake mechanisms that may operate in electroautotrophs are reviewed, aiming at finding possible causes for this low performance. Biomass yields, growth rates and electron uptake rates observed when these microorganisms use chemical electron donors are compared with those typically obtained with electrodes, to explore limitations and advantages inherent to the electroautotrophic metabolism. Also, the factors affecting biofilm development are analysed to show how interfacial interactions condition bacterial adhesion, biofilm growth and electrons uptake. Finally, possible strategies to overcome these limitations are described.

Urine dilution with a synthetic wastewater (Syntho) boosts the electricity production in a bio-electrochemical system powered by un-pretreated human urine.

Human urine can be turned into electricity in bio-electrochemical systems. The acclimation of electro-active bacteria to culture media with increasing urine concentrations has led to raising the obtained current densities, which typically followed a Monod-like evolution profile as a function of urine concentration. However, the acclimation protocol has been so far evaluated using pretreated urine samples (fermented or precipitated), not raw (un-pretreated) urine. We demonstrate that, when un-pretreated urine is used, the microbial adaptation to increasingly concentrated urine leads to a current density profile that does not reach a saturation-like phase, but follows a Han/Levenspiel-type trend (bell-shaped). By diluting un-pretreated urine with a synthetic domestic wastewater (Syntho) up to concentrations matching those of the maximum in the Han/Levenspiel-like current profile (15-20% v/v) it is possible to avoid the drop in the electro-active response, generating anodic current densities as high as 3.6 ± 0.2 A.m-2 (per actual surface area), 35-fold higher than those reached in pure un-pretreated urine.

Analyzing Chemoreceptor Interactions In Vivo with the Trifunctional Cross-Linker TMEA.

Chemoreceptors are dimeric proteins that contain a periplasmic or extracellular domain for ligand binding and an extremely well-conserved cytoplasmic domain for output response control. This latter domain consists in a long α-helical hairpin that forms a four-helix coiled-coil bundle in the dimer. Dimers associate into trimers of dimers in the crystal structure obtained for the cytoplasmic domain of the Escherichia coli serine chemoreceptor, Tsr. Further studies confirmed that this crystal structure reflects the basic unit within the in vivo organization of chemoreceptors. The trimers of dimers form large and stable chemoreceptor clusters in all the prokaryotes that have been studied. Here, we describe the use of TMEA, a trifunctional cross-linker that reacts with sulfhydryl groups, as a tool to study the geometry and dynamics of the interaction between receptors of the same or different types in living cells.

Mutational Replacements at the "Glycine Hinge" of the Escherichia coli Chemoreceptor Tsr Support a Signaling Role for the C-Helix Residue.

Bacterial chemoreceptors are dimeric membrane proteins that transmit signals from a periplasmic ligand-binding domain to the interior of the cells. The highly conserved cytoplasmic domain consists of a long hairpin that in the dimer forms a four-helix coiled-coil bundle. The central region of the bundle couples changes in helix packing that occur in the membrane proximal region to the signaling tip, controlling the activity of an associated histidine kinase. This subdomain contains certain glycine residues that are postulated to form a hinge in chemoreceptors from enteric bacteria and have been largely postulated to play a role in the coupling mechanism, and/or in the formation of higher-order chemoreceptor assemblies. In this work, we directly assessed the importance of the "glycine hinge" by obtaining nonfunctional replacements at each of its positions in the Escherichia coli serine receptor Tsr and characterizing them. Our results indicate that, rather than being essential for proper receptor-receptor interaction, the "glycine hinge" residues are involved in the ability of the receptor to switch between different signaling states. Mainly, the C-helix residue G439 has a key role in shifting the equilibrium toward a kinase-activating conformation. However, we found second-site mutations that restore the chemotactic proficiency of some of the "glycine hinge" mutants, suggesting that a complete hinge is not strictly essential. Rather, glycine residues seem to favor the coupling activity that relies on some other structural features of the central subdomain.

Bacterial chemoreceptors of different length classes signal independently.

Bacterial chemoreceptors sense environmental stimuli and govern cell movement by transmitting the information to the flagellar motors. The highly conserved cytoplasmic domain of chemoreceptors consists in an alpha-helical hairpin that forms in the homodimer a coiled-coil four-helix bundle. Several classes of chemoreceptors that differ in the length of the coiled-coil structure were characterized. Many bacterial species code for chemoreceptors that belong to different classes, but how these receptors are organized and function in the same cell remains an open question. E. coli cells normally code for single class chemoreceptors that form extended arrays based on trimers of dimers interconnected by the coupling protein CheW and the kinase CheA. This structure promotes effective coupling between the different receptors in the modulation of the kinase activity. In this work, we engineered functional derivatives of the Tsr chemoreceptor of E. coli that mimic receptors whose cytoplasmic domain is longer by two heptads. We found that these long Tsr receptors did not efficiently mix with the native receptors and appeared to function independently. Our results suggest that the assembly of membrane-bound receptors of different specificities into mixed clusters is dictated by the length-class to which the receptors belong, ensuring cooperative function only between receptors of the same class.

Stepping stones in the electron transport from cells to electrodes in Geobacter sulfurreducens biofilms.

Geobacter sulfurreducens bacteria grow on biofilms and have the particular ability of using polarized electrodes as the final electron acceptor of their respiratory chain. In these biofilms, electrons are transported through distances of more than 50 µm before reaching the electrode. The way in which electrons are transported across the biofilm matrix through such large distances remains under intense discussion. None of the two mechanisms proposed for explaining the process, electron hopping through outer membrane cytochromes and metallic like conduction through conductive PilA filaments, can account for all the experimental evidence collected so far. Aiming at providing new elements for understanding the basis for electron transport, in this perspective article we present a modelled structure of Geobacter pilus. Its analysis in combination with already existing experimental evidence gives support to the proposal of the "stepping stone" mechanism, in which the combined action of pili and cytochromes allows long range electron transport through the biofilm.

Functional and structural effects of seven-residue deletions on the coiled-coil cytoplasmic domain of a chemoreceptor.

Chemoreceptors transmit signals from the environment to the flagellar motors via a histidine kinase that controls the phosphorylation level of the effector protein CheY. The cytoplasmic domain of chemoreceptors is strongly conserved and consists of a long alpha-helical hairpin that forms, in the dimer, a coiled-coil four-helix bundle. Changes in this domain during evolution are characterized by the presence of seven-residue insertions/deletions located symmetrically with respect to the hairpin turn, suggesting that specific interactions between the helices that form the hairpin are required for function. We assessed the impact of seven-residue deletions on the signalling ability and higher-order organization of the serine chemoreceptor from Escherichia coli. Our results indicate that symmetry alterations between the two branches of the cytoplasmic hairpin seriously compromise chemoreceptor function. Shorter functional versions of Tsr with symmetrical deletions form mixed trimers of dimers when coexpressed with Tar, the aspartate receptor of E. coli. However, Tar function in those cells is impaired, suggesting that the length difference between receptors introduces non-functional distortions into the chemoreceptor cluster. This observation is reinforced by the analysis of coexpression of Tar with chemoreceptors from Rhodobacter sphaeroides that naturally belong to a shorter-length class.

Cross-linking evidence for motional constraints within chemoreceptor trimers of dimers.

Chemotactic behavior in bacteria relies on the sensing ability of large chemoreceptor clusters that are usually located at the cell pole. In Escherichia coli, chemoreceptors exhibit higher-order interactions within those clusters based on a trimer-of-dimers organization. This architecture is conserved in a variety of other bacteria and archaea, implying that receptors in many microorganisms form trimer-of-dimer signaling teams. To gain further insight into the assembly and dynamic behavior of receptor trimers of dimers, we used in vivo cross-linking targeted to cysteine residues at various positions that define six different levels along the cytoplasmic signaling domains of the aspartate and serine chemoreceptors, Tar and Tsr, respectively. We found that the cytoplasmic domains of these receptors are close to each other near the trimer contact region at the cytoplasmic tip and lie farther apart as the receptor dimers approach the cytoplasmic membrane. Tar and Tsr reporter sites within the same or closely adjacent levels readily formed mixed cross-links, whereas reporters located different distances from the tip did not. These findings indicate that there are no significant vertical displacements of one dimer with respect to the others within the trimer unit. Attractant stimuli had no discernible effect on the cross-linking efficiency of any of the reporters tested, but a strong osmotic stimulus reproducibly enhanced cross-linking at most of the reporter sites, indicating that individual dimers may move closer together under this condition.

Disruption of chemoreceptor signalling arrays by high levels of CheW, the receptor-kinase coupling protein.

During chemotactic signalling by Escherichia coli, the small cytoplasmic CheW protein couples the histidine kinase CheA to chemoreceptor control. Although essential for assembly and operation of receptor signalling complexes, CheW in stoichiometric excess disrupts chemotactic behaviour. To explore the mechanism of the CheW excess effect, we measured the physiological consequences of high cellular levels of wild-type CheW and of several CheW variants with reduced or enhanced binding affinities for receptor molecules. We found that high levels of CheW interfered with trimer assembly, prevented CheA activation, blocked cluster formation, disrupted chemotactic ability and elevated receptor methylation levels. The severity of these effects paralleled the receptor-binding affinities of the CheW variants. Because trimer formation may be an obligate step in the assembly of ternary signalling complexes and higher-order receptor arrays, we suggest that all CheW excess effects stem from disruption of trimer assembly. We propose that the CheW-binding sites in receptor dimers overlap their trimer contact sites and that high levels of CheW saturate the receptor-binding sites, preventing trimer assembly. The CheW-trapped receptor dimers seem to be improved substrates for methyltransferase reactions, but cannot activate CheA or assemble into clusters, processes that are essential for chemotactic signalling.