Marine enhanced rock weathering (mERW) is increasingly receiving attention as a marine-based carbon dioxide removal (CDR) technology. The method aims to achieve ocean alkalinity enhancement (OAE) by introducing fast-weathering rocks into coastal systems. The latter is envisioned to act as a large natural biogeochemical reactor, where ambient physical and biological processes can stimulate rock dissolution, thus generating a concomitant alkalinity release and increasing the seawater's capacity to sequester CO2. Olivine has been put forward as the prime candidate mineral for mERW, but at present, no peer-reviewed results are available from larger-scale field studies in coastal areas, so the information about olivine dissolution in marine systems is largely derived from laboratory experiments. As a result, key uncertainties remain concerning the efficiency, CO2 sequestration potential, and impact of olivine-based mERW under relevant field conditions. In this review, we summarize recent research advancements to bridge the gap between existing laboratory results and the real-world environment in which mERW is intended to take place. To this end, we identify the key parameters that govern the dissolution kinetics of olivine in coastal sediments and the associated CO2 sequestration potential, which enable us to identify a number of uncertainties that still remain with respect to the implementation and upscaling of olivine-based ERW, as well as monitoring, reporting, and verification (MRV). From our analysis, we conclude that the current knowledge base is not sufficient to predict the outcome of in situ mERW applications. Particularly, the impact of pore-water saturation on the olivine dissolution rate and the question of the additionality of alkalinity generation remain critical unknowns. To more confidently assess the potential and impact of olivine-based mERW, dedicated pilot studies under field conditions are needed, which should be conducted at a sufficiently large spatial scale and monitored for a long enough time with sufficient temporal resolution. Additionally, our analysis indicates that the specific sediment type of the application site (e.g., cohesive versus permeable) will be a critical factor for olivine-based mERW applications, as it will significantly impact the dissolution rate by influencing the ambient pore-water pH, saturation dynamics, and natural alkalinity generation. Therefore, future field studies should also target different coastal sediment types.@en

Background: Cable bacteria are filamentous members of the Desulfobulbaceae family that are capable of performing centimetre‑scale electron transport in marine and freshwater sediments. This long‑distance electron transport is mediated by a network of parallel conductive fibres embedded in the cell envelope. This fibre network efficiently transports electrical currents along the entire length of the centimetre‑long filament. Recent analyses show that these fibres consist of metalloproteins that harbour a novel nickel‑containing cofactor, which indicates that cable bacteria have evolved a unique form of biological electron transport. This nickel‑dependent conduction mechanism suggests that cable bacteria are strongly dependent on nickel as a biosynthetic resource. Here, we performed a comprehensive comparative genomic analysis of the genes linked to nickel homeostasis. We compared the genome‑encoded adaptation to nickel of cable bacteria to related members of the Desulfobulbaceae family and other members of the Desulfobulbales order. Results: Presently, four closed genomes are available for the monophyletic cable bacteria clade that consists of the genera Candidatus Electrothrix and Candidatus Electronema. To increase the phylogenomic coverage, we additionally generated two closed genomes of cable bacteria: Candidatus Electrothrix gigas strain HY10‑6 and Candidatus Electrothrix antwerpensis strain GW3‑4, which are the first closed genomes of their respective species. Nickel homeostasis genes were identified in a database of 38 cable bacteria genomes (including 6 closed genomes). Gene prevalence was compared to 19 genomes of related strains, residing within the Desulfobulbales order but outside of the cable bacteria clade, revealing several genome‑encoded adaptations to nickel homeostasis in cable bacteria. Phylogenetic analysis indicates that nickel importers, nickel‑binding enzymes and nickel chaperones of cable bacteria are affiliated to organisms outside the Desulfobulbaceae family, with several proteins showing affiliation to organisms outside of the Desulfobacterota phylum. Conspicuously, cable bacteria encode a unique periplasmic nickel export protein RcnA, which possesses a putative cytoplasmic histidine‑rich loop that has been largely expanded compared to RcnA homologs in other organisms. Conclusion: Cable bacteria genomes show a clear genetic adaptation for nickel utilization when compared to closely related genera. This fully aligns with the nickel‑dependent conduction mechanism that is uniquely found in cable bacteria.

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Cable bacteria can reach high densities in coastal sediments, and as a result of their unusual electrogenic lifestyle and intense metabolic activity, exert a major and distinct impact on biogeochemical cycling, both locally in sediments and at the ecosystem level. This appears to be particularly true for seasonally hypoxic systems, but the driving force behind the proliferation of cable bacteria in these systems is not well understood. Moreover, the metabolism of cable bacteria induces strong acid production, which can be buffered through carbonate dissolution in sediments. A strong depletion of alkalinity in the pore water is therefore expected in carbonate-poor sediments. To evaluate the impact of cable bacteria metabolism on sediment geochemistry, we performed field sampling and laboratory sediment incubations in two seasonally hypoxic sites: one carbonate-poor site with low levels of free sulfide in pore water (Yarra Estuary, Australia) and one carbonate-rich site with high free sulfide (Lake Grevelingen, The Netherlands). Active cable bacteria populations were found in both field locations, with higher abundance and activity observed in spring compared to autumn. The sediment incubations tracked the metabolic activity of cable bacteria over time (maximum 84 days), and confirmed the fast development of an electric network (cell doubling time: ∼19 h). These results suggest that cable bacteria are widespread in seasonally hypoxic systems, supporting previous findings. Cable bacteria acidified the sediment by > 1.5 pH units in 6–13 days (differing per site) and their activity accounted for >70% of the oxygen uptake. A clear subsurface accumulation of Fe²⁺ was observed after 8 days of Yarra sediment incubations, indicative of increased FeS dissolution as e-SOx developed. The increased availability of sulfide from FeS dissolution promotes a positive-feedback loop that we infer allowed for a faster development of cable bacteria in the carbonate-poor sediments. A depletion of total alkalinity was observed in the deeper Yarra sediment, whereas, a higher alkalinity efflux was previously observed in the carbonate-rich sediments from Lake Grevelingen. These results suggest a differential pH and alkalinity dynamic due to the interaction between the local carbonate content of the sediment and cable bacteria activity.

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Multicellular cable bacteria display an exceptional form of biological conduction, channeling electric currents across centimeter distances through a regular network of protein fibers embedded in the cell envelope. The fiber conductivity is among the highest recorded for biomaterials, but the underlying mechanism of electron transport remains elusive. Here, we performed detailed characterization of the conductance from room temperature down to liquid helium temperature to attain insight into the mechanism of long-range conduction. A consistent behavior is seen within and across individual filaments. The conductance near room temperature reveals thermally activated behavior, yet with a low activation energy. At cryogenic temperatures, the conductance at moderate electric fields becomes virtually independent of temperature, suggesting that quantum vibrations couple to the charge transport through nuclear tunneling. Our data support an incoherent multistep hopping model within parallel conduction channels with a low activation energy and high transfer efficiency between hopping sites. This model explains the capacity of cable bacteria to transport electrons across centimeter-scale distances, thus illustrating how electric currents can be guided through extremely long supramolecular protein structures.@en

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 (E_a = 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.

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Cable bacteria exhibit a unique metabolism involving long-distance electron transport, significantly impacting elemental cycling in various sediments. These long filamentous bacteria are distributed circumglobally, suggesting an effective mode of dispersal. However, oxygen strongly inhibits their activity, posing a challenge to their dispersal through the water column. We investigated the effective dispersal of marine cable bacteria in a compartmentalised microcosm experiment. Cable bacteria were grown in natural ‘source’ sediment, and their metabolic activity was recorded in autoclaved ‘destination’ cores, which were only accessible through oxygenated seawater. Colonisation occurred over weeks, and destination cores contained only one cable bacterium strain. Filament ‘snippets’ (fragments with a median size of ~15 cells) accumulated in the microcosm water, with about 30% of snippets attached to sediment particles. Snippet release was also observed in situ in a salt marsh creek. This provides a model for the dispersal of cable bacteria through oxygenated water: snippets are formed by filament breakage in the sediment, released into the overlying water and transported with sediment particles that likely offer protection. These insights are informative for broader theories on microbial community assembly and prokaryotic biogeography in marine sediments.@en

Cable bacteria have acquired a unique metabolism, which induces long-distance electron transport along their centimeter-long multicellular filaments. At present, cable bacteria are thought to form a monophyletic clade with two described genera. However, their diversity has not been systematically investigated. To investigate the phylogenetic relationships within the cable bacteria clade, 16S rRNA gene sequences were compiled from literature and public databases (SILVA 138 SSU and NCBI GenBank). These were complemented with novel sequences obtained from natural sediment enrichments across a wide range of salinities (2–34). To enable taxonomic resolution at the species level, we designed a procedure to attain full-length 16S rRNA gene sequences from individual cable bacterium filaments using an optimized nested PCR protocol and Sanger sequencing. The final database contained 1,876 long 16S rRNA gene sequences (≥800 bp) originating from 92 aquatic locations, ranging from polar to tropical regions and from intertidal to deep sea sediments. The resulting phylogenetic tree reveals 90 potential species-level clades (based on a delineation value of 98.7% 16S rRNA gene sequence identity) that reside within six genus-level clusters. Hence, the diversity of cable bacteria appears to be substantially larger than the two genera and 13 species that have been officially named up to now. Particularly brackish environments with strong salinity fluctuations, as well as sediments with low free sulfide concentrations and deep sea sediments harbor a large pool of novel and undescribed cable bacteria taxa.@en

The recent discovery of cable bacteria has greatly expanded the known length scale of biological electron transport, as these multi-cellular bacteria are capable of mediating electrical currents across centimeter-scale distances. To enable such long-range conduction, cable bacteria embed a network of regularly spaced, parallel protein fibers in their cell envelope. These fibers exhibit extraordinary electrical properties for a biological material, including an electrical conductivity that can exceed 100 S cm ⁻¹. Traditionally, long-range electron transport through proteins is described as a multi-step hopping process, in which the individual hopping steps are described by Marcus electron transport theory. Here, we investigate to what extent such a classical hopping model can explain the conductance data recorded for individual cable bacterium filaments. To this end, the conductive fiber network in cable bacteria is modelled as a set of parallel one-dimensional hopping chains. Comparison of model simulated and experimental current(I)/voltage(V) curves, reveals that the charge transport is field-driven rather than concentration-driven, and there is no significant injection barrier between electrodes and filaments. However, the observed high conductivity levels (>100 S cm ⁻¹) can only be reproduced, if we include much longer hopping distances (a > 10 nm) and lower reorganisation energies (λ < 0.2 eV) than conventionally used in electron relay models of protein structures. Overall, our model analysis suggests that the conduction mechanism in cable bacteria is markedly distinct from other known forms of long-range biological electron transport, such as in multi-heme cytochromes.

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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.@en

Many environmentally relevant micro-organisms cannot be cultured, and even with the latest metagenomic approaches, achieving complete genomes for specific target organisms of interest remains a challenge. Cable bacteria provide a prominent example of a microbial ecosystem engineer that is currently unculturable. They occur in low abundance in natural sediments, but due to their capability for long-distance electron transport, they exert a disproportionately large impact on the biogeochemistry of their environment. Current available genomes of marine cable bacteria are highly fragmented and incomplete, hampering the elucidation of their unique electrogenic physiology. Here, we present a metagenomic pipeline that combines Nanopore long-read and Illumina short-read shotgun sequencing. Starting from a clonal enrichment of a cable bacterium, we recovered a circular metagenome-assembled genome (5.09 Mbp in size), which represents a novel cable bacterium species with the proposed name Candidatus Electrothrix scaldis. The closed genome contains 1109 novel identified genes, including key metabolic enzymes not previously described in incomplete genomes of cable bacteria. We examined in detail the factors leading to genome closure. Foremost, native, non-amplified long reads are crucial to resolve the many repetitive regions within the genome of cable bacteria, and by analysing the whole metagenomic assembly, we found that low strain diversity is key for achieving genome closure. The insights and approaches presented here could help achieve genome closure for other keystone micro-organisms present in complex environmental samples at low abundance.

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Cable bacteria are long, filamentous bacteria with a unique metabolism involving centimetre-scale electron transport. They are widespread in the sediment of seasonally hypoxic systems and their metabolic activity stimulates the dissolution of iron sulfides (FeS), releasing large quantities of ferrous iron (Fe²⁺) into the pore water. Upon contact with oxygen, Fe²⁺ oxidation forms a layer of iron(oxyhydr)oxides (FeO_x), which in its turn can oxidize free sulfide (H₂S) and trap phosphorus (P) diffusing upward. The metabolism of cable bacteria could thus prevent the release of H₂S from the sediment and reduce the risk of euxinia, while at the same time modulating P release over seasonal timescales. However, experimental support for this so-called ‘iron firewall hypothesis’ is scarce. Here, we collected natural sediment in a seasonally hypoxic basin in three different seasons. Undisturbed sediment cores were incubated under anoxic conditions and the effluxes of H₂S, dissolved iron (dFe) and phosphate (PO₄³⁻) were monitored for up to 140 days. Cores with recent cable bacterial activity revealed a high stock of sedimentary FeO_x, which delayed the efflux of H₂S for up to 102 days. Our results demonstrate that the iron firewall mechanism could exert an important control on the prevalence of euxinia and regulate the P release in coastal oceans.

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Bacterial cells can vary greatly in size, from a few hundred nanometers to hundreds of micrometers in diameter. Filamentous cable bacteria also display substantial size differences, with filament diameters ranging from 0.4 to 8 µm. We analyzed the genomes of cable bacterium filaments from 11 coastal environments of which the resulting 23 new genomes represent 10 novel species-level clades of Candidatus Electrothrix and two clades that putatively represent novel genus-level diversity. Fluorescence in situ hybridization with a species-level probe showed that large-sized cable bacteria belong to a novel species with the proposed name Ca. Electrothrix gigas. Comparative genome analysis suggests genes that play a role in the construction or functioning of large cable bacteria cells: the genomes of Ca. Electrothrix gigas encode a novel actin-like protein as well as a species-specific gene cluster encoding four putative pilin proteins and a putative type II secretion platform protein, which are not present in other cable bacteria. The novel actin-like protein was also found in a number of other giant bacteria, suggesting there could be a genetic basis for large cell size. This actin-like protein (denoted big bacteria protein, Bbp) may have a function analogous to other actin proteins in cell structure or intracellular transport. We contend that Bbp may help overcome the challenges of diffusion limitation and/or morphological complexity presented by the large cells of Ca. Electrothrix gigas and other giant bacteria. IMPORTANCE In this study, we substantially expand the known diversity of marine cable bacteria and describe cable bacteria with a large diameter as a novel species with the proposed name Candidatus Electrothrix gigas. In the genomes of this species, we identified a gene that encodes a novel actin-like protein [denoted big bacteria protein (Bbp)]. The bbp gene was also found in a number of other giant bacteria, predominantly affiliated to Desulfobacterota and Gammaproteobacteria, indicating that there may be a genetic basis for large cell size. Thus far, mostly structural adaptations of giant bacteria, vacuoles, and other inclusions or organelles have been observed, which are employed to overcome nutrient diffusion limitation in their environment. In analogy to other actin proteins, Bbp could fulfill a structural role in the cell or potentially facilitate intracellular transport.

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Carbon dioxide removal (CDR) technologies at a gigaton scale need to be developed and implemented within the next decades to keep global warming below 1.5 °C. Coastal enhanced silicate weathering is one of the proposed CDR techniques that aims to accelerate the natural process of CO₂-sequestration during marine chemical weathering of silicate minerals. To this end, finely ground rock containing olivine (Mg_xFe_{2− x}SiO₄) could be dispersed in dynamic coastal environments, where local biotic and abiotic factors potentially enhance the weathering process. However, accurate predictions of the olivine dissolution rate and the associated CO₂ sequestration under in situ conditions are currently lacking and ecosystem impacts remain to be assessed. Previously, it has been hypothesized that in situ grain collisions, induced by bed load transport due to currents and waves, could accelerate the in situ chemical weathering of olivine particles. To examine this, we investigated the effects of continuous grain tumbling on olivine dissolution in natural seawater. A 70-day experiment was conducted in which forsterite olivine sand was continuously tumbled in filtered seawater at different rotation speeds, and dissolution rates were measured on a weekly basis. Results showed that continuously tumbled olivine dissolved 8 to 19 times faster compared to stagnant (no rotation) conditions. Olivine dissolution was complete and stoichiometric (except for Ni release), air-seawater CO₂ exchange was not significantly rate limiting, and minimal particle fragmentation and secondary mineral formation were observed. Hence, we infer that olivine weathering was mainly enhanced via advective pore water flushing, which limits saturation effects at the grain scale. Overall, this study provides evidence that ambient physical stresses in coastal environments could enhance marine silicate weathering, which has implications for both the natural silicon cycle as well as the use of enhanced coastal weathering of silicates as a CDR technique.

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Cable bacteria are multicellular sulfide oxidizing bacteria that display a unique metabolism based on long-distance electron transport. Cells in deeper sediment layers perform the sulfide oxidizing half-reaction whereas cells in the surface layers of the sediment perform the oxygen-reducing half-reaction. These half-reactions are coupled via electron transport through a conductive fiber network that runs along the shared cell envelope. Remarkably, only the sulfide oxidizing half-reaction is coupled to biosynthesis and growth whereas the oxygen reducing half-reaction serves to rapidly remove electrons from the conductive fiber network and is not coupled to energy generation and growth. Cells residing in the oxic zone are believed to (temporarily) rely on storage compounds of which polyphosphate (poly-P) is prominently present in cable bacteria. Here we investigate the role of poly-P in the metabolism of cable bacteria within the different redox environments. To this end, we combined nanoscale secondary ion mass spectrometry with dual-stable isotope probing (¹³C-DIC and ¹⁸O-H₂O) to visualize the relationship between growth in the cytoplasm (¹³C-enrichment) and poly-P activity (¹⁸O-enrichment). We found that poly-P was synthesized in almost all cells, as indicated by ¹⁸O enrichment of poly-P granules. Hence, poly-P must have an important function in the metabolism of cable bacteria. Within the oxic zone of the sediment, where little growth is observed, ¹⁸O enrichment in poly-P granules was significantly lower than in the suboxic zone. Thus, both growth and poly-P metabolism appear to be correlated to the redox environment. However, the poly-P metabolism is not coupled to growth in cable bacteria, as many filaments from the suboxic zone showed poly-P activity but did not grow. We hypothesize that within the oxic zone, poly-P is used to protect the cells against oxidative stress and/or as a resource to support motility, while within the suboxic zone, poly-P is involved in the metabolic regulation before cells enter a non-growing stage.

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Eutrophication and global change are increasing the occurrence of seasonal hypoxia (bottom-water oxygen concentration <63 μM) in coastal systems worldwide. In extreme cases, the bottom water can become completely anoxic, allowing sulfide to escape from the sediments and leading to the development of bottom-water euxinia. In seasonally hypoxic coastal basins, electrogenic sulfur oxidation by long, filamentous cable bacteria has been shown to stimulate the formation of an iron oxide layer near the sediment-water interface, while the bottom waters are oxygenated. Upon the development of bottom-water anoxia, this iron oxide “firewall” prevents the sedimentary release of sulfide. Iron oxides also act as an adsorption trap for elements such as arsenic. Arsenic is a toxic trace metal, and its release from sediments can have a negative impact on marine ecosystems. Yet, it is currently unknown how electrogenic sulfur oxidation impacts arsenic cycling in seasonally hypoxic basins. In this study, we presented results from a seasonal field study of an uncontaminated marine lake, complemented with a long-term sediment core incubation experiment, which reveals that cable bacteria have a strong impact on the arsenic cycle in a seasonally hypoxic system. Electrogenic sulfur oxidation significantly modulates the arsenic fluxes over a seasonal time scale by enriching arsenic in the iron oxide layer near the sediment-water interface in the oxic period and pulse-releasing arsenic during the anoxic period. Fluxes as large as 20 μmol m⁻² day⁻¹ were measured, which are comparable to As fluxes reported from highly contaminated sediments. Since cable bacteria are recognized as active components of the microbial community in seasonally hypoxic systems worldwide, this seasonal amplification of arsenic fluxes is likely a widespread phenomenon.

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Fish farming in sea cages is a growing component of the global food industry. A prominent ecosystem impact of this industry is the increase in the downward flux of organic matter, which stimulates anaerobic mineralization and sulfide production in underlying sediments. When free sulfide is released to the overlying water, this can have a toxic effect on local marine ecosystems. The microbially-mediated process of sulfide oxidation has the potential to be an important natural mitigation and prevention strategy that has not been studied in fish farm sediments. We examined the microbial community composition (DNA-based 16S rRNA gene) underneath two active fish farms on the Southwestern coast of Iceland and performed laboratory incubations of resident sediment. Field observations confirmed the strong geochemical impact of fish farming on the sediment (up to 150 m away from cages). Sulfide accumulation was evidenced under the cages congruent with a higher supply of degradable organic matter from the cages. Phylogenetically diverse microbes capable of sulfide detoxification were present in the field sediment as well as in lab incubations, including cable bacteria (Candidatus Electrothrix), which display a unique metabolism based on long-distance electron transport. Microsensor profiling revealed that the activity of cable bacteria did not exert a dominant impact on the geochemistry of fish farm sediment at the time of sampling. However, laboratory incubations that mimic the recovery process during fallowing, revealed successful enrichment of cable bacteria within weeks, with concomitant high sulfur-oxidizing activity. Overall our results give insight into the role of microbially-mediated sulfide detoxification in aquaculture impacted sediments.

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Sulfated glycosaminoglycans (sGAG) are negatively charged extracellular polymeric substances that occur in biofilms from various environments. Yet, it remains unclear whether these polymers are acquired from the external environment or produced by microbes in the biofilm. To resolve this, we analyzed the presence of sGAGs in samples of an acidophilic biofilm collected from Sulfur Cave in Puturosu Mountain (Romania), an environment that is largely inaccessible to contamination. A maximum of 55.16 ± 2.06 μg sGAG-like polymers were recovered per mg of EPS. Enzymatic treatment with chondroitinase ABC resulted in a decrease of the mass of these polymers, suggesting the structure of the recovered sGAG is similar to chondroitin. Subsequent FT-IR analysis of these polymers revealed absorbance bands at 1230 cm⁻¹, 1167 cm⁻¹ and 900 cm⁻¹, indicating a possible presence of polysaccharides and sulfate. Analysis of genomic sequences closely related to those predominant in the acidophilic biofilm, contained genes coding for sulfotransferase (an enzyme needed for the production of sGAG), which supports the hypothesis of microbial synthesis of sGAGs within the biofilm.

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Detailed validation of air quality models is essential, but remains challenging, due to a lack of suitable high-resolution measurement datasets. This is particularly true for pollutants with short-scale spatial variations, such as nitrogen dioxide (NO₂). While street-level air quality model chains can predict concentration gradients at high spatial resolution, measurement campaigns lack the coverage and spatial density required to validate these gradients. Citizen science offers a tool to collect large-scale datasets, but it remains unclear to what extent such data can truly increase model performance. Here we use the passive sampler dataset collected within the large-scale citizen science campaign CurieuzeNeuzen to assess the integrated ATMO-Street street-level air quality model chain. The extensiveness of the dataset (20.000 sampling locations across the densely populated region Flanders, ∼1.5 data points per km²) allowed an in-depth model validation and optimization. We illustrate generic techniques and methods to assess and improve street-level air quality models, and show that considerable model improvement can be achieved, in particular with respect to the correct representation of the small-scale spatial variability of the NO₂-concentrations. After model optimization, the model skill of the ATMO-Street chain significantly increased, passing the FAIRMODE model quality threshold, and thus substantiating its suitability for policy support. More generally, our results reveal how a “deep validation” based on extensive spatial data can substantially improve model performance, thus demonstrating how air quality modelling can benefit from one-off large-scale monitoring campaigns.

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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.

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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.

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