by Gemma Reguera
In a recent post I shared with you how different microbes come together to breathe as one. In some cases, all it takes is the presence of conductive minerals such as magnetite to facilitate the exchange of metabolic electrons between two microbial partners. This allows the team to catalyze a redox reaction (for example, acetate oxidation coupled to nitrate reduction) that each organism could not have been able to achieve individually. The role of conductive minerals in such interspecies electron transfer is a good example of the diverse strategies that microorganisms have evolved to compete for the available electron acceptors, especially those with highest affinity for electrons (the most electropositive ones), as more energy is generated from the reactions.
In marine sediments, for example, oxygen diffuses from the oxic waters into the underlying sediment and is rapidly consumed by microorganisms in the upper region. This limits its availability as a terminal electron acceptor in the deeper layers, which are largely anoxic. As a result, microbes in the anoxic zone rely on alternative electron acceptors, choosing the most electropositive first. This also stratifies the anoxic region of the sediment, with each layer reflecting, from top to bottom, the sequential reduction of nitrate (NO3-), manganese (Mn[IV]), iron (Fe[III], which is often present as insoluble iron oxide minerals) and sulfate (SO4-2). Methanogens, which use the most electronegative electron acceptor (CO2), are usually active in the deepest stratum of the sediment. Interestingly, the reduction of sulfate generates hydrogen sulfide, a toxic gas with the chemical formula H2S. Sulfide production can be high in marine sediments, leading to a characteristic foul smell similar to that of rotten eggs. Unless removed, sulfide can expand from its layer into the oxic layer and poison the respiratory enzymes of oxygen-respiring cells.
Some microbes have evolved mechanisms to detoxify sulfide and/or use it as a substrate for growth. Sulfide can, for example, be oxidized to sulfate or other sulfur compounds by some species even in the presence of oxygen. It can also serve as electron donor for photosynthetic purple and green sulfur bacteria or for nitrate reducers. In addition, sulfide can react strongly with iron and manganese oxides to form iron-sulfur and manganese-sulfur minerals, respectively, which can also be used as terminal electron acceptors. The integration of all these microbial activities in the sediments helps prevent the expansion of the sulfide layer and keep it at millimeter or even centimeters away from the oxic layer. It also creates a harmonic metabolic balance among all the microbes in the sediments, so although each carries out its own reaction they can respond and adapt to sediment disturbances as a whole, integrated system.
In 2010, Nielsen and collaborators reported the surprising finding that the sulfide and oxic layers of marine sediments were somehow interconnected, so that disturbances in the oxic layer were rapidly transmitted to the sulfide layer. Limiting oxygen availability resulted in the rapid expansion of the sulfide layer, and vice versa. In addition, the researchers noted that the pH of the region where oxygen was being reduced increased, as expected from the electrochemical reduction of oxygen, which consumes protons. The responses were so fast that could only be explained if electric currents, rather than diffusible chemical species, coupled the oxidation of sulfide to the reduction of oxygen. At the time, we knew that some bacteria could produce conductive appendages or ‘nanowires’ for long-range electron transfer. We also knew that solid materials generated during the degradation of organic matter such as humic substances could enable the shuttling of electrons between cells in the sediments. Furthermore, conductive minerals such as magnetite could also facilitate the electronic coupling of biogeochemical reactions. However, the available evidence suggested that these mechanisms enabled the transmission of electric currents through nanometer to micrometer distances, rather than the millimeter to centimeter distances that typically separate the oxic and sulfide sediment layers.
In a recent paper published in Nature, Pfeffer and collaborators finally shed some light into the mechanism that could be responsible for the electrical coupling of the reactions taking place in the oxygen and sulfide sediment layers. The authors observed that the sediments were densely colonized with long multicellular filaments of a novel bacterial lineage within the family Desulfobulbaceae. The filaments were so abundant that the researchers calculated a density of 117 m of the bacteria per cm3 sediment. If each filament has an average length of 1 cm, every cm3 of sediment contains more than 1,000 filaments. That’s a lot of filaments packed close together in the sediment! The filaments were fragile and broke off easily during their extraction from the sediments, yet some reached lengths sufficient to span the range (12-15 mm) of spatial separation of the oxic and sulfide sediment layers. Whenever the filament connections were disrupted, for example by inserting filter barriers or by cutting sediment cores transversally, oxygen consumption and the concomitant pH rise diminished or stopped and the sulfide layer expanded. The researchers also showed that replacing the oxic-anoxic interface of the sediment with glass beads made no difference. Thus, the sediment particles were not required for the electrical coupling of the sediment reactions. The team also inserted a tungsten wire into the sediment cores, which short-circuited the electrical connections and uncoupled the reactions of the oxic and sulfide layers. The unique structural and electronic properties of the filaments are suggestive of their involvement in the transmission of electric currents. The outer membrane of the cells, for example, is structured as longitudinal ridges, each containing tubular channels of periplasm linking neighboring cells. Although the outer membrane acts as an insulating sheath, electrostatic force microscopy revealed areas of high polarization along the cellular ridges and cell-cell junctions, suggesting they are electrically charged.
Although all the experimental evidence presented in the Pffefer report is indirect, the results do support a model whereby multicellular filaments function as electric cables to couple the oxidation of sulfide to the reduction of oxygen in the sediments. Still, the centimeter distances that electrons are presumed to travel along the filaments defy our current understanding of biological electron transfer. Thus, more work is needed to demonstrate that these filamentous bacteria do in fact transport electrons along their length and at the rates required to couple the sulfide and oxic layer reactions. It is also important to note that the experiments described in the report did not rule out the involvement, either directly or indirectly, of other microbes and/or other mechanisms of electron transfer. For example, when the filament connections were physically disrupted, other microbial connections (such as nanowire networks) might have been disrupted as well. The model also implies that the metabolic activities of the filaments are stratified along their length, with cells at one end oxidizing sulfide and supplying electrons to the oxygen-reducing cells located in the opposite end of the filament. Other members of the family Desulfobulbaceae family are known to oxidize sulfide with oxygen serving as a terminal electron acceptor, so the same microbe could indeed catalyze both metabolic reactions.
Despite the many questions that still remain unanswered, the available evidence presents a compelling and thought-provoking model of bacterial filaments behaving as living, centimeter-long electric cables. As discoveries go, this one is quite electrifying (pun intended and acknowledged). I simply cannot guess what the answers will be. Regardless, I look forward to the next installment in this series!
Pfeffer C, Larsen S, Song J, Dong M, Besenbacher F, Meyer RL, Kjeldsen KU, Schreiber L, Gorby YA, El-Naggar MY, Leung KM, Schramm A, Risgaard-Petersen N, Nielsen LP (2012). Filamentous bacteria transport electrons over centimetre distances. Nature, 491 (7423), 218−221. PMID 23103872
Nielsen LP, Risgaard-Petersen N, Fossing H, Christensen PB, Sayama M (2010). Electric currents couple spatially separated biogeochemical processes in marine sediment. Nature, 463 (7284), 1071−1074. PMID 20182510
Gemma is assistant professor in the Department of Microbiology and Molecular Genetics, Michigan State University.