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The overlap of microbiology and electrochemistry provides plenty of opportunities for a deeper understanding of the redox biogeochemical cycle of natural-abundant elements (like iron, nitrogen, and sulfur) on Earth. The electroactive microorganisms (EAMs) mediate electron flows outward the cytomembrane via diverse pathways like multiheme cytochromes, bridging an electronic connection between abiotic and biotic reactions. On an environmental level, decades of research on EAMs and the derived subject termed \"electromicrobiology\" provide a rich collection of multidisciplinary knowledge and establish various bioelectrochemical designs for the development of environmental biotechnology. Recent advances suggest that EAMs actually make greater differences on a larger scale, and the metabolism of microbial community and ecological interactions between microbes play a great role in bioremediation processes. In this perspective, we propose the concept of microbial electron transfer network (METN) that demonstrates the \"species-to-species\" interactions further and discuss several key questions ranging from cellular modification to microbiome construction. Future research directions including metabolic flux regulation and microbes-materials interactions are also highlighted to advance understanding of METN for the development of next-generation environmental biotechnology.

Electrotrophy, the growth of microbes on extracellular electron donors, drives important biogeochemical cycles and has practical applications. Studies of Fe(II)-based electrotrophy have provided foundational cytochrome-based mechanistic models for electron transport into cells. Direct electron uptake from other microbial species, Fe(0), or cathodes is of intense interest due to its potential roles in the production and anaerobic oxidation of methane, corrosion, and bioelectrochemical technologies. Other cells or Fe(0) can serve as the sole electron donor supporting the growth of several Geobacter and methanogen strains that are unable to use H2 as an electron donor, providing strong evidence for electrotrophy. Additional evidence for electrotrophy in Geobacter strains and Methanosarcina acetivorans is a requirement for outer-surface c-type cytochromes. However, in most instances claims for electrotrophy in anaerobes are based on indirect inference and the possibility that H2 is actually the electron donor supporting growth has not been rigorously excluded.

Geobacter sulfurreducens is a model microbe for elucidating the mechanisms for extracellular electron transfer in several biogeochemical cycles, bioelectrochemical applications, and microbial metal corrosion. Multiple lines of evidence previously suggested that electrically conductive pili (e-pili) are an essential conduit for long-range extracellular electron transport in G. sulfurreducens. However, it has recently been reported that G. sulfurreducens does not express e-pili and that filaments comprised of multi-heme c-type cytochromes are responsible for long-range electron transport. This possibility was directly investigated by examining cells, rather than filament preparations, with atomic force microscopy. Approximately 90% of the filaments emanating from wild-type cells had a diameter (3 nm) and conductance consistent with previous reports of e-pili harvested from G. sulfurreducens or heterologously expressed in Escherichia coli from the G. sulfurreducens pilin gene. The remaining 10% of filaments had a morphology consistent with filaments comprised of the c-type cytochrome OmcS. A strain expressing a modified pilin gene designed to yield poorly conductive pili expressed 90% filaments with a 3-nm diameter, but greatly reduced conductance, further indicating that the 3-nm diameter conductive filaments in the wild-type strain were e-pili. A strain in which genes for five of the most abundant outer-surface c-type cytochromes, including OmcS, were deleted yielded only 3-nm-diameter filaments with the same conductance as in the wild type. These results demonstrate that e-pili are the most abundant conductive filaments expressed by G. sulfurreducens, consistent with previous functional studies demonstrating the need for e-pili for long-range extracellular electron transfer. IMPORTANCE Electroactive microbes have significant environmental impacts, as well as applications in bioenergy and bioremediation. The composition, function, and even existence of electrically conductive pili (e-pili) has been one of the most contentious areas of investigation in electromicrobiology, in part because e-pili offer a mechanism for long-range electron transport that does not involve the metal cofactors common in much of biological electron transport. This study demonstrates that e-pili are abundant filaments emanating from Geobacter sulfurreducens, which serves as a model for long-range extracellular electron transfer in direct interspecies electron transfer, dissimilatory metal reduction, microbe-electrode exchange, and corrosion caused by direct electron uptake from Fe(0). The methods described in this study provide a simple strategy for evaluating the distribution of conductive filaments throughout the microbial world with an approach that avoids artifactual production and/or enrichment of filaments that may not be physiologically relevant.

Microbially catalyzed corrosion of metals is a substantial economic concern. Aerobic microbes primarily enhance Fe0 oxidation through indirect mechanisms and their impact appears to be limited compared to anaerobic microbes. Several anaerobic mechanisms are known to accelerate Fe0 oxidation. Microbes can consume H2 abiotically generated from the oxidation of Fe0. Microbial H2 removal makes continued Fe0 oxidation more thermodynamically favorable. Extracellular hydrogenases further accelerate Fe0 oxidation. Organic electron shuttles such as flavins, phenazines, and possibly humic substances may replace H2 as the electron carrier between Fe0 and cells. Direct Fe0-to-microbe electron transfer is also possible. Which of these anaerobic mechanisms predominates in model pure culture isolates is typically poorly documented because of a lack of functional genetic studies. Microbial mechanisms for Fe0 oxidation may also apply to some other metals. An ultimate goal of microbial metal corrosion research is to develop molecular tools to diagnose the occurrence, mechanisms, and rates of metal corrosion to guide the implementation of the most effective mitigation strategies. A systems biology approach that includes innovative isolation and characterization methods, as well as functional genomic investigations, will be required in order to identify the diagnostic features to be gleaned from meta-omic analysis of corroding materials. A better understanding of microbial metal corrosion mechanisms is expected to lead to new corrosion mitigation strategies. The understanding of the corrosion microbiome is clearly in its infancy, but interdisciplinary electrochemical, microbiological, and molecular tools are available to make rapid progress in this field.

Intrinsically conductive protein nanowires, microbially produced from inexpensive, renewable feedstocks, are a sustainable alternative to traditional nanowire electronic materials, which require high energy inputs and hazardous conditions/chemicals for fabrication and can be highly toxic. Pilin-based nanowires can be tailored for specific functions via the design of synthetic pilin genes to tune wire conductivity or introduce novel functionalities. Other microbially produced nanowire options for electronics may include cytochrome wires, curli fibers, and the conductive fibers of cable bacteria. Proof-of-concept protein nanowire electronics that have been successfully demonstrated include biomedical sensors, neuromorphic devices, and a device that generates electricity from ambient humidity. Further development of applications will require interdisciplinary teams of engineers, biophysicists, and synthetic biologists.

The concept that anaerobic microorganisms can directly accept electrons from Fe(0) has been controversial because direct metal-microbe electron transfer has previously only been indirectly inferred. Fe(0) oxidation was studied with Geobacter sulfurreducens strain ACL, an autotrophic strain that was previously shown to grow with electrons derived from a graphite cathode as the sole electron donor. Strain ACL grew with Fe(0) as the sole electron donor and fumarate as the electron acceptor. However, it appeared that at least a portion of the electron transfer was via H2 produced nonenzymatically from the oxidation of Fe(0) to Fe(II). H2, which accumulated in abiotic controls, was consumed during the growth of strain ACL, the cells were predominately planktonic, and genes for the uptake hydrogenase were highly expressed. Strain ACLHF was constructed to prevent growth on H2 or formate by deleting the genes for the uptake of hydrogenase and formate dehydrogenases from strain ACL. Strain ACLHF also grew with Fe(0) as the sole electron donor, but H2 accumulated in the culture, and cells heavily colonized Fe(0) surfaces with no visible planktonic growth. Transcriptomics suggested that the outer surface c-type cytochromes OmcS and OmcZ were important during growth of strain ACLHF on Fe(0). Strain ACLHF did not grow on Fe(0) if the gene for either of these cytochromes was deleted. The specific attachment of strain ACLHF to Fe(0), coupled with requirements for known extracellular electrical contacts, suggest that direct metal-microbe electron transfer is the most likely option for Fe(0) serving as an electron donor.IMPORTANCE The anaerobic corrosion of iron structures is expensive to repair and can be a safety and environmental concern. It has been known for over 100 years that the presence of anaerobic respiratory microorganisms can accelerate iron corrosion. Multiple studies have suggested that there are sulfate reducers, methanogens, and acetogens that can directly accept electrons from Fe(0) to support sulfate or carbon dioxide reduction. However, all of the strains studied can also use H2 as an electron donor for growth, which is known to be abiotically produced from Fe(0). Furthermore, no proteins definitely shown to function as extracellular electrical contacts with Fe(0) were identified. The studies described here demonstrate that direct electron transfer from Fe(0) can support anaerobic respiration. They also map out a simple genetic approach to the study of iron corrosion mechanisms in other microorganisms. A better understanding of how microorganisms promote iron corrosion is expected to lead to the development of strategies that can help reduce adverse impacts from this process.