A Cross-System Comparison of Dark Carbon Fixation in Coastal Sediments

Dark carbon fixation (DCF) by chemoautotrophic microorganisms can sustain food webs in the seafloor by local production of organic matter independent of photosynthesis. The process has received considerable attention in deep sea systems, such as hydrothermal vents, but the regulation, depth distribution, and global importance of coastal sedimentary DCF have not been systematically investigated. Here we surveyed eight coastal sediments by means of stable isotope probing (13C-DIC) combined with bacterial biomarkers (phospholipid-derived fatty acids) and compiled additional rates from literature into a global database. DCF rates in coastal sediments range from 0.07 to 36.30 mmol C m−2 day−1, and there is a linear relation between DCF and water depth. The CO2 fixation ratio (DCF/CO2 respired) also shows a trend with water depth, decreasing from 0.09 in nearshore environments to 0.04 in continental shelf sediments. Five types of depth distributions of chemoautotrophic activity are identified based on the mode of pore water transport (advective, bioturbated, and diffusive) and the dominant pathway of microbial sulfur oxidation. Extrapolated to the global coastal ocean, we estimate a DCF rate of 0.04 to 0.06 Pg C year−1, which is less than previous estimates based on indirect measurements (0.15 Pg C year−1), but remains substantially higher than the global DCF rate at deep sea hydrothermal vents (0.001–0.002 Pg C year−1).BT/Environmental Biotechnolog

Spatial variability in macrofaunal diet composition and grazing pressure on microphytobenthos in intertidal areas

Microphytobenthos forms an important part of the diet of macrofauna (macrozoobenthos) in many intertidal ecosystems. It is unclear, however, whether the dependence of macrofauna on microphytobenthos varies spatially within and among tidal systems. We aim (1) to assess the spatial variability in the importance of microphytobenthos in the diet of macrofauna (i.e., between and within two tidal basins and as function of elevation), (2) to quantify grazing pressure of the macrofaunal community on different potential food sources (microphytobenthos, phytoplankton and terrestrial organic material) for several sites in two tidal basins and (3) to compare microphytobenthic production and summer/autumn grazing of the total macrofaunal community and grazing pressure per feeding type, with potential microphytobenthic production estimated from rates in early spring, when grazing was low. Using a natural stable isotope approach, we identified microphytobenthos as a more important food source for macrofauna than phytoplankton and terrestrial organic material. Microphytobenthos dependency differed between tidal basins for the genera Bathyporeia (sand digger shrimp), Macoma (Baltic tellin), and Peringia (mudsnail) and for sampled individuals of all genera combined, and did not vary as function of elevation. We showed that macrofaunal grazing on microphytobenthos is quantitatively important and, in some cases, approached microphytobenthic production rates in early spring. No positive relation between microphytobenthic production in early spring and macrofaunal grazing in summer/autumn was observed. This suggests that the studied consumer-resource interactions are coupled on a larger spatial scale (i.e., mesoscale, ≈10 to 100 km), rather than the fine (mm to m) scale.Environmental Fluid MechanicsBT/Environmental Biotechnolog

Enhanced Laterally Resolved ToF-SIMS and AFM Imaging of the Electrically Conductive Structures in Cable Bacteria

Cable bacteria are electroactive bacteria that form a long, linear chain of ridged cylindrical cells. These filamentous bacteria conduct centimeter-scale long-range electron transport through parallel, interconnected conductive pathways of which the detailed chemical and electrical properties are still unclear. Here, we combine time-of-flight secondary-ion mass spectrometry (ToF-SIMS) and atomic force microscopy (AFM) to investigate the structure and composition of this naturally occurring electrical network. The enhanced lateral resolution achieved allows differentiation between the cell body and the cell-cell junctions that contain a conspicuous cartwheel structure. Three ToF-SIMS modes were compared in the study of so-called fiber sheaths (i.e., the cell material that remains after the removal of cytoplasm and membranes, and which embeds the electrical network). Among these, fast imaging delayed extraction (FI-DE) was found to balance lateral and mass resolution, thus yielding the following multiple benefits in the study of structure-composition relations in cable bacteria: (i) it enables the separate study of the cell body and cell-cell junctions; (ii) by combining FI-DE with in situ AFM, the depth of Ni-containing protein - key in the electrical transport - is determined with greater precision; and (iii) this combination prevents contamination, which is possible when using an ex situ AFM. Our results imply that the interconnects in extracted fiber sheaths are either damaged during extraction, or that their composition is different from fibers, or both. From a more general analytical perspective, the proposed methodology of ToF-SIMS in the FI-DE mode combined with in situ AFM holds great promise for studying the chemical structure of other biological systems.BT/Environmental Biotechnolog

The organo-metal-like nature of long-range conduction in cable bacteria

Cable bacteria are filamentous, multicellular microorganisms that display an exceptional form of biological electron transport across centimeter-scale distances. Currents are guided through a network of nickel-containing protein fibers within the cell envelope. Still, the mechanism of long-range conduction remains unresolved. Here, we characterize the conductance of the fiber network under dry and wet, physiologically relevant, conditions. Our data reveal that the fiber conductivity is high (median value: 27 S cm−1; range: 2 to 564 S cm−1), does not show any redox signature, has a low thermal activation energy (Ea = 69 ± 23 meV), and is not affected by humidity or the presence of ions. These features set the nickel-based conduction mechanism in cable bacteria apart from other known forms of biological electron transport. As such, conduction resembles that of an organic semi-metal with a high charge carrier density. Our observation that biochemistry can synthesize an organo-metal-like structure opens the way for novel bio-based electronic technologies.BT/Environmental Biotechnolog

Multi-wavelength Raman microscopy of nickel-based electron transport in cable bacteria

Cable bacteria embed a network of conductive protein fibers in their cell envelope that efficiently guides electron transport over distances spanning up to several centimeters. This form of long-distance electron transport is unique in biology and is mediated by a metalloprotein with a sulfur-coordinated nickel (Ni) cofactor. However, the molecular structure of this cofactor remains presently unknown. Here, we applied multi-wavelength Raman microscopy to identify cell compounds linked to the unique cable bacterium physiology, combined with stable isotope labeling, and orientation-dependent and ultralow-frequency Raman microscopy to gain insight into the structure and organization of this novel Ni-cofactor. Raman spectra of native cable bacterium filaments reveal vibrational modes originating from cytochromes, polyphosphate granules, proteins, as well as the Ni-cofactor. After selective extraction of the conductive fiber network from the cell envelope, the Raman spectrum becomes simpler, and primarily retains vibrational modes associated with the Ni-cofactor. These Ni-cofactor modes exhibit intense Raman scattering as well as a strong orientation-dependent response. The signal intensity is particularly elevated when the polarization of incident laser light is parallel to the direction of the conductive fibers. This orientation dependence allows to selectively identify the modes that are associated with the Ni-cofactor. We identified 13 such modes, some of which display strong Raman signals across the entire range of applied wavelengths (405–1,064 nm). Assignment of vibrational modes, supported by stable isotope labeling, suggest that the structure of the Ni-cofactor shares a resemblance with that of nickel bis(1,2-dithiolene) complexes. Overall, our results indicate that cable bacteria have evolved a unique cofactor structure that does not resemble any of the known Ni-cofactors in biology.BT/Environmental Biotechnolog

A highly conductive fibre network enables centimetre-scale electron transport in multicellular cable bacteria

Biological electron transport is classically thought to occur over nanometre distances, yet recent studies suggest that electrical currents can run along centimetre-long cable bacteria. The phenomenon remains elusive, however, as currents have not been directly measured, nor have the conductive structures been identified. Here we demonstrate that cable bacteria conduct electrons over centimetre distances via highly conductive fibres embedded in the cell envelope. Direct electrode measurements reveal nanoampere currents in intact filaments up to 10.1 mm long (>2000 adjacent cells). A network of parallel periplasmic fibres displays a high conductivity (up to 79 S cm-1), explaining currents measured through intact filaments. Conductance rapidly declines upon exposure to air, but remains stable under vacuum, demonstrating that charge transfer is electronic rather than ionic. Our finding of a biological structure that efficiently guides electrical currents over long distances greatly expands the paradigm of biological charge transport and could enable new bio-electronic applications.BT/Environmental BiotechnologyBN/Bertus Beaumont LabQN/van der Zant La

Efficient long-range conduction in cable bacteria through nickel protein wires

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.BT/Environmental Biotechnolog

Division of labor and growth during electrical cooperation in multicellular cable bacteria

Multicellularity is a key evolutionary innovation, leading to coordinated activity and resource sharing among cells, which generally occurs via the physical exchange of chemical compounds. However, filamentous cable bacteria display a unique metabolism in which redox transformations in distant cells are coupled via long-distance electron transport rather than an exchange of chemicals. This challenges our understanding of organismal functioning, as the link among electron transfer, metabolism, energy conservation, and filament growth in cable bacteria remains enigmatic. Here, we show that cells within individual filaments of cable bacteria display a remarkable dichotomy in biosynthesis that coincides with redox zonation. Nanoscale secondary ion mass spectrometry combined with 13C (bicarbonate and propionate) and 15N-ammonia isotope labeling reveals that cells performing sulfide oxidation in deeper anoxic horizons have a high assimilation rate, whereas cells performing oxygen reduction in the oxic zone show very little or no label uptake. Accordingly, oxygen reduction appears to merely function as a mechanism to quickly dispense of electrons with little to no energy conservation, while biosynthesis and growth are restricted to sulfide-respiring cells. Still, cells can immediately switch roles when redox conditions change, and show no differentiation, which suggests that the “community service” performed by the cells in the oxic zone is only temporary. Overall, our data reveal a division of labor and electrical cooperation among cells that has not been seen previously in multicellular organisms.BT/Environmental Biotechnolog

Long-distance electron transport in individual, living cable bacteria

Electron transport within living cells is essential for energy conservation in all respiring and photosynthetic organisms. While a few bacteria transport electrons over micrometer distances to their surroundings, filaments of cable bacteria are hypothesized to conduct electric currents over centimeter distances. We used resonance Raman microscopy to analyze cytochrome redox states in living cable bacteria. Cable-bacteria filaments were placed in microscope chambers with sulfide as electron source and oxygen as electron sink at opposite ends. Along individual filaments a gradient in cytochrome redox potential was detected, which immediately broke down upon removal of oxygen or laser cutting of the filaments. Without access to oxygen, a rapid shift toward more reduced cytochromes was observed, as electrons were no longer drained from the filament but accumulated in the cellular cytochromes. These results provide direct evidence for long-distance electron transport in living multicellular bacteria.BT/Environmental Biotechnolog

Cell Cycle, Filament Growth and Synchronized Cell Division in Multicellular Cable Bacteria

Cable bacteria are multicellular, Gram-negative filamentous bacteria that display a unique division of metabolic labor between cells. Cells in deeper sediment layers are oxidizing sulfide, while cells in the surface layers of the sediment are reducing oxygen. The electrical coupling of these two redox half reactions is ensured via long-distance electron transport through a network of conductive fibers that run in the shared cell envelope of the centimeter-long filament. Here we investigate how this unique electrogenic metabolism is linked to filament growth and cell division. Combining dual-label stable isotope probing (13C and 15N), nanoscale secondary ion mass spectrometry, fluorescence microscopy and genome analysis, we find that the cell cycle of cable bacteria cells is highly comparable to that of other, single-celled Gram-negative bacteria. However, the timing of cell growth and division appears to be tightly and uniquely controlled by long-distance electron transport, as cell division within an individual filament shows a remarkable synchronicity that extends over a millimeter length scale. To explain this, we propose the “oxygen pacemaker” model in which a filament only grows when performing long-distance transport, and the latter is only possible when a filament has access to oxygen so it can discharge electrons from its internal electrical network.BT/Environmental Biotechnolog