Microbial Extracellular Electron Transfer is a Far-Out Metabolism

Nov. 15, 2019

According to Nobel Prize winning biochemist Albert Szent-Györgyi: “Life is nothing but an electron looking for a place to rest.” Although life is more complex than a single electron, Szent-Györgyi’s quote emphasizes the importance of energy and metabolism in all living systems. The many different methods that cells use to harvest and process energy from their environment constitutes much of the diversity we observe in the microbial world. That metabolic diversity includes microorganisms that can “breathe” rocks and make electricity.
 
All cells rely on oxidation and reduction (redox) reactions to help produce energy. Electrons are released from an electron donor (oxidation), and are received by the electron acceptor (reduction). Energy is harvested from electrons as they are transferred from one chemical to another; our cells oxidize carbon from the food we eat and reduce the oxygen we breathe. Intracellular enzymes reduce oxygen, which can enter the cell, and captures energy to power ATP production.

However, oxygen was not in the atmosphere during the days of primordial soup, so microbes used other respiration strategies, including reducing electron acceptors that are physically outside their cells. Some microbes “breathe” solid iron (III) oxide-containing rocks that cannot enter the cell. Electrons must move outside of the cell to reduce the extracellular electron acceptor in the process of extracellular electron transfer (EET). Scientists initially discovered microorganisms capable of EET nearly 30 years ago and have since invented ground-breaking applications for these fascinating microorganisms, including replacing rocks with electrodes to produce electricity.

The Search for Extracellular Electron Transfer (EET)

The field of EET sparked from the search for an explanation of 2 separate environmental observations. 

One of these observations was that manganese oxide minerals “disappear” from the sediments of Lake Oneida in New York, but only during the summer. The solid manganese oxide was reduced to soluble manganese when the bottom of the lake became anaerobic during periods of warmer temperature. Although scientists reported this “disappearance” in multiple marine and freshwater ecosystems, chemistry and geology could not explain how this manganese was reduced.

The other observation was the mysterious oxidation of acetate in Potomac River sediments. In subsurface sediments, degradation of complex carbon produces acetate. Acetate must be metabolized to CO2 to complete the carbon cycle of the ecosystem. The zone in the soil where acetate oxidation occurs did not include a known electron acceptor, but did contain rust (iron (III) oxide).

How was manganese oxide being reduced in Lake Oneida? What was the electron acceptor for acetate oxidation in the Potomac River? The then-existing models of energy flow were insufficient, and redox reactions were unbalanced. Where were the electrons going? Microbial reduction of mineral oxides could account for some of these missing electrons. The problem was, there were no microorganisms described at that time that could support this hypothesis.

To find microorganism(s) capable of metal reduction, researchers followed in the footsteps of microbiology forebears, Martinus W. Beijerinck and Sergey Winogradsky. Two labs isolated microorganisms capable of EET around the same time using enrichment cultures with mineral oxides as electron acceptors. Shewanella oneidensis was isolated from Lake Oneida sediments while oxidizing succinate and reducing manganese oxide. Geobacter metallireducens was isolated from freshwater Potomac River sediments while oxidizing acetate and reducing iron oxide. Discovering these microorganisms was only the first step in solving the mysterious observations.

Two Model Microbes Are Better than One: Multiple Mechanisms for EET

Although microbes explaining the Lake Oneida and Potomac River redox reactions had been found, the mystery of how electrons cross the cell membrane to reach extracellular mineral oxides remained. Shewanella and Geobacter species are Gram-negative bacteria and thus they have 2 lipid bilayers surrounding the cell, the inner and the outer membrane. Moving electrons across a lipid bilayer is like trying to conduct electricity using rubber: it stinks.
 
One clue was the characteristic color of G. metallireducens and S. oneidensis colonies: pink. Just like hemoglobin in our red blood cells, heme-containing cytochrome proteins are also pink. Cytochromes are capable of various redox reactions throughout biology as either membrane-bound or soluble proteins. Decades of research have shown numerous cytochromes are involved in transporting electrons from the inner membrane, across the periplasm, and through the outer membrane to reduce the extracellular electron acceptor.

(Left) Shewanella oneidensis MR-1 colonies grown on LB agar. (Right) Geobacter sulfurreducens PCA colonies grown on minimal medium with acetate as the electron donor and fumarate as the electron acceptor. The salmon-pink color of Shewanella and red color of Geobacter species is due to large amounts of cytochromes produced by these bacteria.
Source: Photo courtesy of B. Conley

 
Having 2 model systems for EET highlights how microorganisms have evolved different metabolic processes around a core mechanism. One example of different solutions to the same problem of reaching out and touching a distant electron acceptor are conductive cellular extensions made by Shewanella and Geobacter species.

Shewanella ‘reaches out’ to electron acceptors using outer membrane vesicles that are linked together into outer membrane extensions. The extensions contain cytochromes full of electrons, and these electrons are delivered to the extracellular acceptor. Shewanella responds to its environment by expanding, branching and retracting the extensions from the cell potentially to increase the chance of interacting with an extracellular electron acceptor.

Instead of membrane extensions, Geobacter species use conductive protein filaments as cellular extensions, but the exact composition of these filaments is an area of active research. The protein filaments were identified as conductive pili, or e-pili (previously referred to as nanowires). Pili are extracellular protein filaments characterized for their role in DNA uptake, adhesion, biofilm formation and motility. Recent analysis of Geobacter species conductive protein filaments shows they are composed of micrometer long chains of cytochromes. Whatever the composition of conductive filaments, these studies have opened the door to potential applications of interfacing conductive living sensors with electrical devices to report environmental conditions.

Cellular extensions are 1 of the 3 general ways we currently know of for electrons to move from a cell to an extracellular acceptor. Shewanella and Geobacter species both use these general mechanisms in slightly different ways, as summarized in the table below. The diversity of EET mechanisms are amazing because their evolution occurred under unique selective pressures. Geobacter and Shewanella species are evolutionarily distinct and reside in discrete environmental niches. Despite differences in cellular physiology and community structure, the use of EET by both species has reinforced the importance of continued characterization of the wide variety of metabolic processes of the microbial world. 

 
  Geobacter spp. Shewanella spp.
Natural environment Anaerobic sediments, typically iron (III) rich Marine and freshwater sediment and water, sometimes in association with aquatic animals.
Phylogeny δ-Proteobacteria γ-Proteobacteria
Pathway  Multiple pathways dependent on extracellular electron acceptor  Conserved single pathway to reduce all extracellular electron acceptors
Biofilms on electrodes Multilayered and active at 10 μm away from electron acceptor Single layer of cells
Shuttle production Soluble cytochrome (PgcA) Flavin mononucleotide (FMN), redox active molecule 
Cellular extensions e-pili and/or cytochrome chains   Outer membrane extensions 
 
General mechanisms of extracellular electron transport. (A) Geobacter and Shewanella species use direct contact of extracellular reductases to the electron acceptor. (B) Secretion and/or utilization of a soluble molecule or protein to carry electrons from the cell to the electron acceptor. Shewanella spp. secrete their own electron shuttle, while Listeria spp. use shuttles available in the environmental milieu. Geobacter spp. secrete and use a soluble cytochrome to shuttle electrons. (C) Production of protein (Geobacter spp.) or proteinaceous membrane extensions (Shewanella spp.) to carry electrons from the cell to the electron acceptor.
Source: Image courtesy B. Conley, created on Inkscape.


EET Exists in Multiple Environments Including the Mammalian Host

EET may allow microorganisms to ditch electrons extracellularly to maintain redox balance within the cell when preferential electron acceptors are absent, such as in an anaerobic gut. Experiments with Listeria monocytogenes mutants unable to perform EET show a growth defect compared to wild-type in anaerobic conditions both in vitro and in vivo. The food-borne pathogen can perform EET, but isn’t found in mineral oxide-rich environments like Shewanella and Geobacter species, demonstrating that EET can be used in a wide variety of environments.

Multiple Gram-positive bacteria have been shown to perform EET, but how do electrons leave the cell in microbes surrounded by a thick layer of peptidoglycan and a single plasma membrane? Instead of cytochromes, L. monocytogenes uses protein-bound and environmental flavins to shuttle electrons to the extracellular electron acceptor. Homologs to the pathway in L. monocytogenes are encoded in many other Firmicutes that perform EET including Enterococcus faecalis, Clostridium perfringens, Bacillus subtilis, Lactococcus lactis and Thermoanaerobacter ethanolicus.

There are other Firmicutes, such as the thermophile Thermincola ferriacetica, that perform EET and do not encode homologs to the L. monocytogenes pathway. The genome of T. ferriacetica is predicted to be enriched in cytochromes which may be involved in performing EET. How cytochromes are anchored and arranged in the cell wall of T. ferriacetica remains to be determined.

Why Study Extracellular Electron Transfer?

Initial characterizations of EET-capable microorganisms focused on Geobacter and Shewanella species. Building upon this knowledge, we now know there are Acidobacteria, Actinobacteria, Deferribacteres and even Archaea (Crenarchaeota and Euryarchaeota) that perform EET.

EET is not limited to reduction and can be performed in the opposite direction; oxidizing extracellular electron donors and moving electrons into the cell. These iron oxidizing bacteria encounter the same solubility hurdles as iron reducing bacteria, electron uptake must occur outside of the cell because the resulting mineral oxide is typically solid and would create a rock inside the cell. Because EET can occur in both redox “directions,” microorganisms can transfer electrons directly from cell to cell. One bacterium serves as the electron acceptor and the other serves as the electron donor. 

Over the past 30 years, collaborative teams of engineers, chemists and biologists have explored applications of EET. Bioremediation and sensing of toxic minerals such as uranium, arsenic and chromium by EET-capable microorganisms has been investigated with varying success. Replacing mineral oxides with an electrode creates modest amounts of electricity and has been used to power deep sea devices. Bioenergy can also be produced as a byproduct in wastewater treatment

Reduction of mineral oxides alters the dynamics of mineral cycling in the environment, as observed in Lake Oneida and Potomac River sediments. On a larger scale, EET helped shape our planet, as observed in the geological rock record. Learning about the microorganisms that shaped Earth may even guide us in learning about other planets. As humans look up into the stars, we turn to microbial metabolism to understand what else might be out there. The surface of Mars is covered in iron oxide minerals. Perhaps electric microbes are up on Mars searching for a place to rest their electrons. 
Banded iron formation from Soudan Underground Mine State Park, Minnesota. Red lines are iron (III) oxide minerals. Black lines are typically magnetite (iron (II,III) oxide mineral). In the ancient ocean, biotic and abiotic oxidation of iron (II) caused precipitation of solid iron (III) oxide minerals to the sea floor. EET capable microorganisms reduced iron (III) oxide to iron (II) containing magnetite. Repeated oxidation and reduction of iron leads to the layering observed.
Source: Photo courtesy Chi Ho Chan at the University of Minnesota
 
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Author: Bridget Conley

Bridget Conley
Bridget Conley is a PhD candidate at the University of Minnesota studying the who, why and how microbes breathe solid minerals. She enjoys learning about cool science and sharing that passion with others through various outreach activities. Bridget is the ASM Young Ambassador to Minnesota, and you can connect with her on Twitter or LinkedIn.