Shewanella Decolorationis S12 Research Articles

2D covalent organic framework/reduced graphene oxide hybrid hydrogel for simultaneous power generation and organic contamination degradation in microbial fuel cell

Microbial fuel cells (MFCs) are promising devices that convert chemical energy into electrical energy via extracellular electron transfer between bacteria and electrodes. However, inefficient extracellular electron transfers and bacteria/electrode interactions often limit their performance. Herein, we demonstrate a hybrid hydrogel assembled by a 2D covalent organic framework (COF) and reduced graphene oxide (rGO) nanosheets as anode materials for MFCs. The 2D COF nanosheets effectively prevent the stacking of rGO nanosheets, resulting in the hybrid hydrogel with a high specific surface area (313.2 m2/g), abundant mesopores and macropores, and high electrical conductivity (492 S/m). The COF material also brings many quinone groups as redox mediators to facilitate electron transfer between bacteria and electrodes. The COF/rGO hybrid hydrogel enhances the growth of Shewanella decolorationis S12 cells, even under the influence of different organic dyes. Dense biofilms are formed on the hybrid hydrogel's surface and inside its macropores, further improving extracellular electron transfers. Assembled MFCs using COF/rGO anodes deliver a maximum power density of 905.1 mW/m2 and can operate for 600 h. They also enable simultaneous power generation and fast organic contamination degradation in electrolytes. This work shows the potential of using 2D nanoarchitectures to promote high-performance MFCs.

SERS combined with the difference in bacterial extracellular electron transfer ability to distinguish Shewanella

Shewanella plays an important role in geochemical cycle, biological corrosion, bioremediation and bioenergy. The development of methods for identifying Shewanella can provide technical support for its rapid screening, in-depth research into its extracellular respiratory mechanism and its application in ecological environment remediation. As a tool for microbial classification, identification and detection, Surface-enhanced Raman scattering (SERS) has high feasibility and application potential. In this work, bio-synthesized silver nanoparticles (AgNPs) were used as SERS substrates to effectively distinguish different types of Shewanella bacteria based on the difference in bacterial extracellular electron transfer (EET) ability. AgNPs were combined with the analyzed bacteria to prepare “Bacteria-AgNPs” SERS samples, which can strongly enhance the Raman signal of the target bacteria and reliably obtain spatial information of different molecular functional groups of each bacteria. Our developed approach can effectively distinguish between non-metal reducing and metal-reducing bacteria, and can further distinguish the three subspecies of Shewanella (Shewanella oneidensis MR-1, Shewanella decolorationis S12, and Shewanella putrefaciens SP200) at the genus and species level. The Raman signal enhancement is presumably caused by the excitation of local surface plasma (LSP) and the enhancement of surrounding electric field. Therefore, our developed method can achieve interspecific and intraspecies discrimination of bacteria. The proposed method can be extended to distinguish other metal-reducing bacteria, and the novel SERS active substrates can be developed for practical applications.

Soil Amoebae Affect Iron and Chromium Reduction through Preferential Predation between Two Metal-Reducing Bacteria.

Soil protists are essential but often overlooked in soil and could impact microbially driven element cycling in natural ecosystems. However, how protists influence heavy metal cycling in soil remains poorly understood. In this study, we used a model protist, Dictyostelium discoideum, to explore the effect of interactions between soil amoeba and metal-reducing bacteria on the reduction of soil Fe(III) and Cr(VI). We found that D. discoideum could preferentially prey on the Fe(III)-reducing bacterium Shewanella decolorationis S12 and significantly decrease its biomass. Surprisingly, this predation pressure also stimulated the activity of a single S. decolorationis S12 bacterium to reduce Fe(III) by enhancing the content of electron-transfer protein cyt c, intracellular ATP synthesis, and reactive oxygen species (e.g., H2O2). We also found that D. discoideum could not prey on the Cr(VI)-reducing bacterium Brevibacillus laterosporus. In contrast, B. laterosporus became edible to amoebae in the presence of S. decolorationis S12, and their Cr(VI) reduction ability decreased under amoeba predation pressure. This study provides direct evidence that protists can affect the Cr and Fe cycling via the elective predation pressure on the metal-reducing bacteria, broadening our horizons of predation of protists on soil metal cycling.

Molecular mechanism of zero valent iron-enhanced microbial azo reduction

Zero valent iron (ZVI)–microbe technology has an increasing application on the removal of organic pollution, yet the molecular mechanism of microbe respond to ZVI is still a mystery. Here, we established a successive ZVI-enhanced microbial system to remove azo dye (a typical organic pollutant) by Shewanella decolorationis S12 (S. decolorationis S12, an effective azo dye degradation bacterium) and examined the gene expression time course (10, 30, 60, and 120 min) by whole genome transcriptional analysis. The addition of ZVI to the microbial degradation system increases the rate of azo reduction from ~60% to over 99% in 16 h reaction, suggesting the synergistic effect of ZVI and S12 on azo dye degradation. Comparing with the treatment without ZVI, less filamentous cells were observed in ZVI treated system, and approximately 8% genes affiliated with 10 different gene expression profiles in S. decolorationis S12 were significantly changed in 120 min during the ZVI-enhanced azo reduction. Intriguingly, MarR transcriptional factor might play a vital role in regulating ZVI-enhanced azo reduction in the aspect of energy production, iron homeostasis, and detoxification. Further investigation showed that the induced [Ni–Fe] H2ase genes (hyaABCDEF) and azoreductase genes (mtrABC-omcA) contributed to ZVI-enhanced energy production, while the reduced iron uptake (hmuVCB and feoAB), induced sulfate assimilation (cysPTWA) and cysteine biosynthesis (cysM) related genes were essential to iron homeostasis and detoxification. This study disentangles underlying mechanisms of ZVI-enhanced organic pollution biotreatment in S. decolorationis S12.

Function-Oriented Graphene Quantum Dots Probe for Single Cell in situ Sorting of Active Microorganisms in Environmental Samples.

Functional microorganisms play a vital role in removing environmental pollutants because of their diverse metabolic capability. Herein, a function-oriented fluorescence resonance energy transfer (FRET)-based graphene quantum dots (GQDs-M) probe was developed for the specific identification and accurate sorting of azo-degrading functional bacteria in the original location of environmental samples for large-scale culturing. First, nitrogen-doped GQDs (GQDs-N) were synthesized using a bottom-up strategy. Then, a GQDs-M probe was synthesized based on bonding FRET-based GQDs-N to an azo dye, methyl red, and the quenched fluorescence was recovered upon cleavage of the azo bond. Bioimaging confirmed the specific recognition capability of GQDs-M upon incubation with the target bacteria or environmental samples. It is suggested that the estimation of environmental functional microbial populations based on bioimaging will be a new method for rapid preliminary assessment of environmental pollution levels. In combination with a visual single-cell sorter, the target bacteria in the environmental samples could be intuitively screened at the single-cell level in 17 bacterial strains, including the positive control Shewanella decolorationis S12, and were isolated from environmental samples. All of these showed an azo degradation function, indicating the high accuracy of the single-cell sorting strategy using the GQDs-M. Furthermore, among the bacteria isolated, two strains of Bacillus pacificus and Bacillus wiedmannii showed double and triple degradation efficiency for methyl red compared to the positive control (strain S12). This strategy will have good application prospects for finding new species or high-activity species of specific functional bacteria.

Lack of Periplasmic Non-heme Protein SorA Increases Shewanella decolorationis Current Generation.

Bacterial extracellular electron transport (EET) plays an important role in many natural and engineering processes. Some periplasmic non-heme redox proteins usually coexist with c-type cytochromes (CTCs) during the EET process. However, in contrast to CTCs, little is known about the roles of these non-heme redox proteins in EET. In this study, the transcriptome of Shewanella decolorationis S12 showed that the gene encoding a periplasmic sulfite dehydrogenase molybdenum-binding subunit SorA was significantly up-regulated during electrode respiration in microbial fuel cells (MFCs) compared with that during azo-dye reduction. The maximum current density of MFCs catalyzed by a mutant strain lacking SorA (ΔsorA) was 25% higher than that of wild strain S12 (20 vs. 16 μA/cm2). Both biofilm formation and the current generation of the anodic biofilms were increased by the disruption of sorA, which suggests that the existence of SorA in S. decolorationis S12 inhibits electrode respiration. In contrast, disruption of sorA had no effect on respiration by S. decolorationis S12 with oxygen, fumarate, azo dye, or ferric citrate as electron acceptors. This is the first report of the specific effect of a periplasmic non-heme redox protein on EET to electrode and provides novel information for enhancing bacterial current generation.

FRET-based fluorescent nanoprobe platform for sorting of active microorganisms by functional properties

Fluorescence-activated cell sorting (FACS) has rarely been applied to screening of microorganisms because of poor detection resolution, which is compromised by poor stability, toxicity, or interference from background fluorescence of the fluorescence sensors used. Here, a fluorescence-based rapid high-throughput cell sorting method was first developed using a fluorescence resonance energy transfer (FRET) fluorescent nanoprobe NP-RA, which was constructed by coating a silica nanoparticle with Rhodamine B and methyl-red (an azo dye). Rhodamine B (inner layer) is the FRET donor and methyl-red (outer layer) is the acceptor. This ready-to-use NP-RA is non-fluorescent, but fluoresces once the outer layer is degraded by microorganisms. In our experiment, NP-RA was ultrasensitive to model strain Shewanella decolorationis S12, showing a broad detection range from 8.0 cfu/mL to 8.7 × 108 cfu/mL under confocal laser scanning microscopy, and from 1.1 × 107 to 9.36 × 108 cfu/mL under a fluorometer. In addition, NP-RA bioimaging can clearly identify other azo-respiring cells in the microbial community, including Bosea thiooxidans DSM 9653 and Lysinibacillus pakistanensis NCCP-54. Furthermore, the fluorescent probe NP-RA is compatible with downstream FACS so that azo-respiring cells can be rapidly sorted out directly from an artificial microbial community. To our knowledge, no fluorescent nanoprobe has yet been designed for tracking and sorting azo-respiration functional microorganisms.

Effects of Flavin-Goethite Interaction on Goethite Reduction by Shewanella decolorationis S12.

Flavin mononucleotide (FMN) and riboflavin are structurally similar flavins, except for the presence of a phosphate group on the FMN molecule. They are used by a variety of electroactive bacteria as extracellular electron shuttles in microbial Fe reduction and inevitably interact with Fe (hydr)oxides in the extracellular environment. It is currently unknown whether flavin/Fe (hydr)oxide interaction interferes with extracellular electron transfer (EET) to the mineral surface. In this study, we found that the goethite reduction rate was lower when mediated by FMN than by RF, suggesting that FMN was less effective in shuttling electrons between cells and minerals. Nevertheless, the phosphate group did not prevent the FMN molecule from accepting electrons from bacterial cells and transferring electrons to the mineral. Results of adsorption experiment, attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy, and bacterial attachment trend analyses showed that FMN exhibited strong adsorption on goethite surface by forming phosphate inner-sphere complex, which prevented bacterial cells from approaching goethite. Therefore, the interaction between FMN and goethite surface may increase the distance of electron transfer from bacterial cells to goethite and result in lower EET efficiency in comparison to those mediated by riboflavin. To our knowledge, these data reveal for the first time that the interaction between flavin and Fe (hydr)oxide affect flavin-mediated electron transfer to mineral surface and add a new dimension to our understanding of flavin-mediated microbial Fe reduction processes.

Goethite Hinders Azo Dye Bioreduction by Blocking Terminal Reductive Sites on the Outer Membrane of Shewanella decolorationis S12.

Iron (hydr)oxides are the most ubiquitous Fe(III)-containing minerals in the near-surface environments and can regulate organic pollutant biotransformation by participating in bacterial extracellular electron transfer under anaerobic conditions. Mechanisms described so far are based on their redox properties in bacterial extracellular respiration. Here, we find that goethite, a typical iron (hydr)oxide, inhibits the bioreduction of different polar azo dyes by Shewanella decolorationis S12 not through electron competition, but by the contact of its surface Fe(III) with the bacterial outer surface. Through the combined results of attenuated total reflectance (ATR) Fourier transform infrared spectroscopy, two-dimensional correlation spectroscopy, and confocal laser scanning microscope, we found that the outer membrane proteins MtrC and OmcA of strain S12 are key binding sites for goethite surface. Meanwhile, they were identified as the important reductive terminals for azo dyes. These results suggest that goethite may block the terminal reductive sites of azo dyes on the bacterial outer membrane to inhibit their bioreduction. This discovered role of goethite in bioreduction provides new insight into the microbial transformation processes of organic pollutants in iron (hydr)oxide-containing environments.

Functional response of sediment bacterial community to iron-reducing bioaugmentation with Shewanella decolorationis S12.

Bioaugmentation with exogenously functional microbes is a widely used technology in bioengineering and environmental remediation. Generally, the colonization of inoculated bacteria is considered to be the determining factor in technical success. However, increasing reports have shown that bioaugmentation was still effective when the colonization of inoculated bacteria was unsuccessful. Here, an augmentation study with iron-reducing bacteria (IRB, Shewanella decolorationis S12) was conducted in Fe(II)-poor sediments to elucidate the role of exogenously inoculated bacteria for bioaugmentation performance. The results showed that a sufficient amount of IRB inputs enhanced the iron reduction in bioaugmented sediments, even though the exogenous IRB did not colonize after the beginning of the experiment (less than 1% at day 3). The iron reduction function responded to stimulation of the indigenous IRB community such as Clostridium, Cupriavidus, Fervidicella, and Acinetobacter, which comprised less than 1% in the initial sediments. Moreover, compared with microbial community in control sediment, more positive correlations between OTUs were observed for that in S12-added sediments upon network analysis. The pH and oxidation-reduction potential of sediment were found to be the predominant factors shaping the iron-reducing microbial communities. It meant that exogenous IRB successfully trigged functional community via altering microenvironment by the inoculated bacteria. Overall, this study provides a new insight into the understanding of the role of single strain addition in iron-reducing bioaugmentation.

Mechanism of dyeing technique of Xiang-yun-sha and bioaugmentation of its coating mud with iron reducing bacteria

The dyeing technique of Xiang-yun-sha , with a history of more than 600 years, is a kind of unique hand-made dyeing process that combines both plant dyeing (immersed with Juliang extract) and mineral dyeing (mud coating). It has been included in the national intangible cultural heritage. However, the scientific mechanism of this heritage has not been systematically explained. In this study, the composition and structure of Juliang were analyzed with ionizing-spray high-resolution mass spectrometry and infrared spectroscopy, and the interaction mechanism of the Juliang with silk protein was explored. The mud-coating process was explained from the molecular level through a chemical simulation method, and the maintenance effect of microbes on characteristics of river mud was examined. Results showed that there are phenol and quinone of catechin condensed tannin in the Juliang extract, and they were combined with silk protein peptide bonds through multi-sites hydrogen bonds. The gial pigments on the silk surface after exposure further reacted with Fe(II) in the mud to form soluble condensed tannin complex, and then this complex was rapidly oxidized to form black precipitate containing Fe(III). Eventually, lustrous dark brown was formed on sun side, brown on the other side. Further, it was found that microbial iron reduction played a key role on maintaining the mud properties, because the addition of Shewanella decolorationis S12 with iron reducing function can significantly improve the dyeing performance of unqualified mud. This study provides reliable scientific support to protect inheritance of dyeing technique and promote the standardized production of the industry.

Electricity Generation by Shewanella decolorationis S12 without Cytochrome c.

Bacterial extracellular electron transfer (EET) plays a key role in various natural and engineering processes. Outer membrane c-type cytochromes (OMCs) are considered to be essential in bacterial EET. However, most bacteria do not have OMCs but have redox proteins other than OMCs in their extracellular polymeric substances of biofilms. We hypothesized that these extracellular non-cytochrome c proteins (ENCP) could contribute to EET, especially with the facilitation of electron mediators. This study compared the electrode respiring capacity of wild type Shewanella decolorationis S12 and an OMC-deficient mutant. Although the OMC-deficient mutant was incapable in direct electricity generation in normal cultivation, it regained electricity generation capacity (26% of the wide type) with the aid of extracellular electron mediator (riboflavin). Further bioelectrochemistry and X-ray photoelectron spectroscopy analysis suggested that the ENCP, such as proteins with Fe–S cluster, may participate in the falvin-mediated EET. The results highlighted an important and direct role of the ENCP, generated by either electricigens or other microbes, in natural microbial EET process with the facilitation of electron mediators.

Electron acceptor redox potential globally regulates transcriptomic profiling in Shewanella decolorationis S12.

Electron acceptor redox potential (EARP) was presumed to be a determining factor for microbial metabolism in many natural and engineered processes. However, little is known about the potentially global effects of EARP on bacteria. In this study, we compared the physiological and transcriptomic properties of Shewanella decolorationis S12 respiring with different EARPs in microbial electrochemical systems to avoid the effects caused by the other physicochemical properties of real electron acceptor. Results showed that the metabolic activities of strain S12 were nonlinear responses to EARP. The tricarboxylic acid cycle for central carbon metabolism was down-regulated while glyoxylate shunt was up-regulated at 0.8 V compared to 0.2 and −0.2 V, which suggested that EARP is an important but not the only determinant for metabolic pathways of strain S12. Moreover, few cytochrome c genes were differentially expressed at different EARPs. The energy intensive flagella assembly and assimilatory sulfur metabolism pathways were significantly enriched at 0.8 V, which suggested strain S12 had stronger electrokinesis behavior and oxidative stress-response at high EARP. This study provides the first global information of EARP regulations on microbial metabolism, which will be helpful for understanding microorganism respiration.

Characteristics and Kinetic Analysis of AQS Transformation and Microbial Goethite Reduction:Insight into "Redox mediator-Microbe-Iron oxide" Interaction Process.

The characteristics and kinetics of redox transformation of a redox mediator, anthraquinone-2-sulfonate (AQS), during microbial goethite reduction by Shewanella decolorationis S12, a dissimilatory iron reduction bacterium (DIRB), were investigated to provide insights into “redox mediator-iron oxide” interaction in the presence of DIRB. Two pre-incubation reaction systems of the “strain S12- goethite” and the “strain S12-AQS” were used to investigate the dynamics of goethite reduction and AQS redox transformation. Results show that the concentrations of goethite and redox mediator, and the inoculation cell density all affect the characteristics of microbial goethite reduction, kinetic transformation between oxidized and reduced species of the redox mediator. Both abiotic and biotic reactions and their coupling regulate the kinetic process for “Quinone-Iron” interaction in the presence of DIRB. Our results provide some new insights into the characteristics and mechanisms of interaction among “quinone-DIRB- goethite” under biotic/abiotic driven.

From red to green: the propidium iodide-permeable membrane of Shewanella decolorationis S12 is repairable

Viability is a common issue of concern in almost all microbial processes. Fluorescence-based assays are extensively used in microbial viability assessment, especially for mixed-species samples or biofilms. Propidium iodide (PI) is the most frequently used fluorescence indicator for cell viability based on the membrane permeability. Our results showed that the accumulation of succinate from fumarate respiration could induce PI-permeability in Shewanella decolorationis biofilm cells. Confocal laser scanning microscope further showed that the PI-permeable membrane could be repaired in situ when the extracellular succinate was eliminated by switching fumarate respiration to electrode respiration. Simultaneously with the membrane repair, the electrode respiring capacity of the originally PI-permeable cells was recovered. Agar-colony counts suggested that a major portion of the repaired cells were viable but nonculturable (VBNC). The results evidenced that S. decolorationis S12 has the capacity to repair PI-permeable membranes which suggests a reevaluation of the fate and function of the PI-permeable bacteria and expanded our knowledge on the flexibility of bacterial survival status in harsh environments.

Kinetics of Chemical and Microbial Iron Reduction Along an Inundation Gradient in a Tidal Marsh of the Min River Estuary, Southeastern China

Chemical (via buffered ascorbate-citrate) and microbial (via Shewanella decolorationis S12) iron reduction kinetics in the surficial tidal marsh sediments of the Min River Estuary were compared at six locations along the inundation gradient. A reactive continuum model was successfully applied to describe the kinetic results. Along the inundation gradient, an enhanced tidal inundation frequency towards the sea resulted in changes in the pH, Eh, salinity, as well as the abundances and fractions of reactive Fe(II) and Fe(III). Significant differences were observed between the chemical and microbial iron reductions among the six locations. Larger reducible Fe(III) pools, higher initial rates, and lower mineral heterogeneity were observed in chemical assays when compared to microbial assays. The microbial and chemical reducible Fe(III) as well as their initial reduction rates generally decreased along the inundation gradient. The chemical mineral heterogeneity gradually increased along the inundation gradient, while the microbial mineral heterogeneities were not significantly different at the six locations. Consequently, a higher iron reduction potential was observed in the uplands when compared to that in the lowlands. The result highlights the role of tidal hydrology and topography in iron reduction and suggest that both the pool and mineral heterogeneities of highly reactive Fe(III) should be considered in iron biogeochemical investigations.

Characterization of the enhancement of zero valent iron on microbial azo reduction.

BackgroundThe microbial method for the treatment of azo dye is promising, but the reduction of azo dye is the rate-limiting step. Zero valent iron (Fe0) can enhance microbial azo reduction, but the interactions between microbes and Fe0 and the potential mechanisms of enhancement remain unclear. Here, Shewanella decolorationis S12, a typical azo-reducing bacterium, was used to characterize the enhancement of Fe0 on microbial decolorization.ResultsThe results indicated that anaerobic iron corrosion was a key inorganic chemical process for the enhancement of Fe0 on microbial azo reduction, in which OH−, H2, and Fe2+ were produced. Once Fe0 was added to the microbial azo reduction system, the proper pH for microbial azo reduction was maintained by OH−, and H2 served as the favored electron donor for azo respiration. Subsequently, the bacterial biomass yield and viability significantly increased. Following the corrosion of Fe0, nanometer-scale Fe precipitates were adsorbed onto cell surfaces and even accumulated inside cells as observed by transmission electron microscope energy dispersive spectroscopy (TEM-EDS).ConclusionsA conceptual model for Fe0-assisted azo dye reduction by strain S12 was established to explain the interactions between microbes and Fe0 and the potential mechanisms of enhancement. This model indicates that the enhancement of microbial azo reduction in the presence of Fe0 is mainly due to the stimulation of microbial growth and activity by supplementation with elemental iron and H2 as an additional electron donor. This study has expanded our knowledge of the enhancement of microbial azo reduction by Fe0 and laid a foundation for the development of Fe0-microbial integrated azo dye wastewater treatment technology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0419-3) contains supplementary material, which is available to authorized users.

The characteristics and two-step reaction model of p-nitroacetophenone biodegradation mediated by Shewanella decolorationis S12 and electron shuttle in the presence/absence of goethite

The current study mainly focused on the biodegradation process of p-nitroacetophenone (NP) in the presence and absence of goethite mediated by iron-reducing microbe (Shewanella decolorationis S12) and electron shuttle. The results showed that introduction of electron shuttle could obviously lead to an accumulation of biodegradation intermediate, especially in reaction systems containing high content of electron shuttle in the absence of goethite. Goethite could enhance the degree and rate of NP biodegradation. The microbial reductively generated Fe(II) played an active role in the biodegradation process. The relationship between the concentrations of biodegradation end product and the reaction times could be fitted by a consecutive reaction model with correlation coefficients (adjusted R2) in the range from 0.9241 to 0.9831 during the biodegradation stage from the beginning to about 250 h of incubation. However, during the subsequent biodegradation stages, in the presence and absence of goethite, transitions from the consecutive reaction model to zero-order reaction model and from the consecutive reaction model to exponential growth reaction model were observed, respectively. The newly proposed two-step reaction model will help understand the mechanism of the biodegradation process of nitroaromatic compounds and related pollutants.

Draft Genome Sequence of Shewanella decolorationis S12, a Dye-Degrading Bacterium Isolated from a Wastewater Treatment Plant

Shewanella decolorationis is a valuable microorganism for degrading diverse synthetic textile dyes. Here, we present an annotated draft genome sequence of S. decolorationis S12, which contains 4,219 protein-coding genes and 86 structural RNAs. This information regarding the genetic basis of this bacterium can greatly advance our understanding of the physiology of this species.

The Mechanism, Thermodynamic and Kinetic Characteristics of the Microbial Reduction of Goethite Mediated by Anthraquinone-2-Sulfonate

The microbial reduction process of goethite by Shewanella decolorationis S12 was evaluated. The results showed the electron shuttle, anthraquinone-2-sulfonate (AQS), could enhance the microbial reduction. The thermodynamic and kinetic characteristics of goethite reduction by microorganisms were influenced by AQS, concentrations of iron oxide, and electron donor. Transformation between oxidized and reduced species of the electron shuttle during the microbial reduction could be newly noticed. Two interactive steps, biotic and abiotic, were involved in the microbial reduction of Fe (III) oxide mediated by electron shuttle.