Substrate competition over 320 days maintains extracellular electron transfer and parallel genomic evolution in Shewanella oneidensis MR-1

Complete uranium bioreduction in 48 hours: Synergistic electron transfer in a synthetic microbial consortium

Enhancement of hexavalent chromium reduction by Shewanella oneidensis MR-1 in presence of copper nanoparticles via stimulating bacterial extracellular electron transfer and environmental adaptability

Disentangling the roles of free and cytochrome-bound flavins in extracellular electron transport from Shewanella oneidensis MR-1

In addition to its natural environmental significance, the process of extracellular electron transfer (EET) can be exploited at electrode interfaces to enable microbial electrochemical technologies for renewable energy recovery, biofuel production, bioremediation, and wastewater treatment. Success in these applications hinges on a better understanding of the electron transfer pathways that link electrochemically active microbes, such as Shewanella oneidensis MR-1, to electrodes. Here, we report electrochemical measurements that reveal both flavin-dependent and flavin-independent EET pathways from S. oneidensis MR-1. Significantly, differential pulse voltammetry captured the redox signatures of both free and cytochrome-bound flavins simultaneously in one experimental system for the first time, allowing us to compare their relative contributions while studying the impact of cell-removal, flavin addition, different culture conditions, and a mutant disrupted in flavin secretion. Our results indicate that endogenous flavins accelerate EET from Shewanella to carbon cloth electrodes primarily as cytochrome-bound cofactors, rather than free soluble shuttles, thereby supporting one of the two debated models of Shewanella EET. Our measurements also highlight the extent to which different electrode materials can impact detection of microbial redox signatures. This communication motivates additional structural studies to study the binding location and precise interaction of flavins at cytochrome-electrode interfaces.

Shewanella oneidensis MR-1 is one of the model microorganisms used for extracellular electron transfer. In this study, to elucidate the capability and the relevant metabolic processes of S. oneidensis MR-1 involved in an electron transferring environment, we employed genome-scale modelling to model the necessary metabolic states and flux adjustments for electricity generation in the cytochrome c-based direct electron transfer (DET) mode, the NADH-linked mediated electron transfer (MET) mode and a presumable mixed mode comprising DET and flavin secretion. These are difficult to develop experimentally. The results showed that the microbe had the potential to achieve current outputs of up to 2.610 A/gDW in the DET mode, 2.189 A/gDW in the MET mode and 2.197 A/gDW in the mixed mode. Compared with the DET mode, which relied on cytochrome c oxidase (EC: 1.1.1.2) to mediate the electron transfer, the MET mode was mainly dependent on two routes, catalysed by isocitrate dehydrogenase (NAD) (EC: 1.1.1.4) and NAD transhydrogenase, for the computed high current density value. In the mixed mode, whereas the cytochrome c-based DET accounted for most of the computed maximum current output value, the two flavins combined, riboflavin and FMN, played a much less important role in the probed current value. Shewanella oneidensis MR-1 has the potential to sustain a high extracellular electron transfer rate similarly to Geobacter sulfurreducens, but relies on different intracellular mechanisms. Various levels of electron transfer rates are achieved by different combinations of metabolic pathways. Flavins can significantly degenerate the maximum electricity generation capability of the cell and the biomass formation, and thus should be avoided in order to achieve a high coulombic efficiency.

Multiheme cytochromes play a central role in extracellular electron transfer, a process that allows microorganisms to sustain their metabolism with external electron acceptors or donors. In Shewanella oneidensis MR-1, the decaheme cytochromes OmcA and MtrC show functional specificity for interaction with soluble and insoluble redox partners. In this work, the capacity of extracellular electron transfer by mutant variants of S. oneidensis MR-1 OmcA was investigated. The results show that amino acid mutations can affect protein stability and alter the redox properties of the protein, without affecting the ability to perform extracellular electron transfer to methyl orange dye or a poised electrode. The results also show that there is a good correlation between the reduction of the dye and the current generated at the electrode for most but not all mutants. This observation opens the door for investigations of the molecular mechanisms of interaction with different electron acceptors to tailor these surface exposed cytochromes towards specific bio-based applications.

Shewanella oneidensis MR-1 is capable of extracellular electron transfer (EET) and hence has attracted considerable attention. The EET pathways mainly consist of c-type cytochromes, along with some other proteins involved in electron transfer processes. By whole genome study and protein interactions inquisition, we constructed a large-scale electron transfer network containing 2276 interactions among 454 electron transfer related proteins in S. oneidensis MR-1. Using the k-shell decomposition method, we identified and analyzed distinct parts of the electron transfer network. We found that there was a negative correlation between the ks (k-shell values) and the average DR_100 (disordered regions per 100 amino acids) in every shell, which suggested that disordered regions of proteins played an important role during the formation and extension of the electron transfer network. Furthermore, proteins in the top three shells of the network are mainly located in the cytoplasm and inner membrane; these proteins can be responsible for transfer of electrons into the quinone pool in a wide variety of environmental conditions. In most of the other shells, proteins are broadly located throughout the five cellular compartments (cytoplasm, inner membrane, periplasm, outer membrane, and extracellular), which ensures the important EET ability of S. oneidensis MR-1. Specifically, the fourth shell was responsible for EET and the c-type cytochromes in the remaining shells of the electron transfer network were involved in aiding EET. Taken together, these results show that there are distinct functional parts in the electron transfer network of S. oneidensis MR-1, and the EET processes could achieve high efficiency through cooperation through such an electron transfer network.

Shewanella oneidensis MR-1, as a model electroactive microorganism (EAM) for extracellular electron transfer (EET) study, plays a key role in advancing practical applications of bio-electrochemical systems (BES). Efficient genome-level manipulation tools are vital to promote EET efficiency; thus, a powerful and rapid base editing toolbox in S. oneidensis MR-1 is developed. Firstly a CRISPR/dCas9-AID base editor that shows a relatively narrow editing window restricted to the "-20 to -16" range upstream of the protospacer adjacent motif (PAM) is constructed. Cas9 is also confined by its native PAM requirement, NGG. Then to expand the editable scope, the sgRNA and the Cas-protein to broaden the editing window to "-22 to -9" upstream of the PAM are engineered, and the PAM field to NNN is opened up. Consequently, the coverage of the editable gene is expanded from 89% to nearly 100% in S. oneidensis MR-1. This whole genome-scale cytidine deaminase-based base editing toolbox (WGcBE) is applied to regulate the cell length and the biofilm morphology, which enhances the EET efficiency by 6.7-fold. WGcBE enables an efficient deactivation of genes with full genome coverage, which would contribute to the in-depth and multi-faceted EET study in Shewanella.

Shewanella oneidensis MR-1 is quickly becoming a synthetic biology workhorse for bioelectrochemical technologies due to a high level of understanding of its interaction with electrodes. Transmembrane electron transfer via the Mtr pathway has been well characterized, however, the role of NADH dehydrogenases in feeding electrons to Mtr has been only minimally studied in S. oneidensis MR-1. Four NADH dehydrogenases are encoded in the genome, suggesting significant metabolic flexibility in oxidizing NADH under a variety of conditions. A strain lacking the two dehydrogenases essential for aerobic growth exhibited a severe growth defect with an anode (+0.4 VSHE) or Fe(III)-NTA as the terminal electron acceptor. Our study reveals that the same NADH dehydrogenase complexes are utilized under oxic conditions or with a high potential anode. Our study also supports the previously indicated importance of pyruvate dehydrogenase activity in producing NADH during anerobic lactate metabolism. Understanding the role of NADH in extracellular electron transfer may help improve biosensors and give insight into other applications for bioelectrochemical systems.

Electron acceptor dependence of electron shuttle secretion and extracellular electron transfer by Shewanella oneidensis MR-1

Biochar facilitates ferrihydrite reduction by Shewanella oneidensis MR-1 through stimulating the secretion of extracellular polymeric substances

Microbial electrochemical technologies, also known as bioelectrochemical systems (BESs), including microbial fuel cells (MFCs), microbial electrolysis cells (MECs), microbial electrosynthesis (MES) etc., serve as a diverse platform that combines waste treatment and energy or chemical production utilizing microbial catalytic reactions. MFCs, as a green and sustainable technology enabling simultaneous wastewater treatment and bioelectricity harvest, have attracted extensive attention in recent decades. Different strategies have been implemented to enhance extracellular electron transfer of the well-known electroactive strain, Shewanella oneidensis MR-1, to improve the performance of MFCs. Electroactive biofilms play essential roles in determining the power output of MFCs. To engineer the electroactive biofilm formation of Shewanella oneidensis MR-1, a c-di-GMP biosynthesis gene ydeH was heterologously overexpressed in S. oneidensis MR-1, generating an engineered strain in which the expression of ydeH is under the control of IPTG-inducible promoter, and a strain in which ydeH is under the control of a constitutive promoter. Such engineered Shewanella strains had significantly enhanced biofilm formation and bioelectricity generation. The MFCs inoculated with these engineered strains accomplished a maximum power density of 167.6 ± 3.6 mW/m2, which was ~2.8 times of that achieved by the wild-type MR-1 (61.0 ± 1.9 mW/m2). In addition, a synthetic microbial consortium containing exoelectrogen Shewanella oneidensis MR-1 and riboflavin producing strain, Bacillus subtilis RH33, was rationally designed and successfully constructed, enabling a stable, multiple cycles of MFC operations for more than 500 hours. The maximum power density of MFCs with this synthetic microbial consortium was 277.4 mW/m2, which was 4.9 times of that with MR-1 (56.9 mW/m2) and 40.2 times of RH33 (6.9 mW/m2), separately. At the same time, the Coulombic efficiency of the synthetic microbial consortium (5.6%) was higher than MR-1 (4.1%) and RH33 (2.3%). In the synthetic microbial consortium, it was found that both mediated and direct electron transfer efficiency were enhanced in mixed-culture. By exchanging the anolyte of MR-1 and RH33, it was confirmed that the improved MFC performance with the synthetic microbial consortium was because MR-1 could efficiently utilize the high concentration of riboflavin produced by RH33. Furthermore, external resistance is one of the important factors that affect the performance of MFCs. Bioelectrochemical and biofilm characterization was conducted for Shewanella oneidensis MR-1 inoculated MFCs with 250 Ω, 500 Ω, 2 kΩ, 6 kΩ, and 22 kΩ resistors. In overall, smaller external resistance resulted in higher maximum power density and more riboflavin secretion. Maximum power density of 136.8 ± 3.1 mW/m2 was achieved when MFCs were operated with 500 Ω resistor, which was 3.7 times of that with 22 kΩ resistor. Electrochemical impedance spectra analysis verified an increased internal resistance along with higher external resistance. Meanwhile more biofilm mass and extracellular polymer substances were confirmed on MFC anode with higher external resistance.

Detecting the structural evolution of passive film induced by Shewanella oneidensis MR-1 using aberration-corrected TEM

Involvement of c-type cytochrome CymA in the electron transfer of anaerobic nitrobenzene reduction by Shewanella oneidensis MR-1

Microbial electrochemical technologies (METs) have emerged in recent years as a promising alternative green source of energy, with microbes consuming organic matter to produce energy or valuable byproducts. It is the ability of performing extracellular electron transfer that allows these microbes to exchange electrons with an electrode in these systems. The low levels of current achieved have been the limiting factor for the large-scale application of METs. Shewanella oneidensis MR-1 is one of the most studied electroactive organisms regarding extracellular electron transfer, and it has been shown that biofilm formation is a key factor for current generation. The transcription factor bolA has been identified as a central player in biofilm formation in other organisms, with its overexpression leading to increased biofilm. In this work we explore the effect of this gene in biofilm formation and current production by S. oneidensis MR-1. Our results demonstrate that an increased biofilm formation and consequent current generation was achieved by the overexpression of this gene. This information is crucial to optimize electroactive organisms toward their practical application in METs.