Dissimilatory nitrate reduction by a freshwater cable bacterium.

Cable bacteria (CB) are filamentous Desulfobulbaceae that split the energy-conserving reaction of sulfide oxidation into two half reactions occurring in distinct cells. CB can use nitrate, but the reduction pathway is unknown, making it difficult to assess their direct impact on the N-cycle. Here we show that the freshwater cable bacterium Ca. Electronema sp. GS performs dissimilatory nitrate reduction to ammonium (DNRA). 15NO3--amended sediment with Ca. Electronema sp. GS showed higher rates of DNRA and nitrite production than sediment without Ca. Electronema sp. GS. Electron flux from sulfide oxidation, inferred from electric potential (EP) measurements, matched the electron flux needed to drive CB-mediated nitrate reduction to nitrite and ammonium. Ca. Electronema sp. GS expressed a complete nap operon for periplasmic nitrate reduction to nitrite, and a putative octaheme cytochrome c (pOCC), whose involvement in nitrite reduction to ammonium remains to be verified. Phylogenetic analysis suggests that the capacity for DNRA was acquired in multiple events through horizontal gene transfer from different organisms, before CB split into different salinity niches. The architecture of the nitrate reduction system suggests absence of energy conservation through oxidative phosphorylation, indicating that CB primarily conserve energy through the half reaction of sulfide oxidation.

Cable bacteria at oxygen-releasing roots of aquatic plants: a widespread and diverse plant-microbe association.

Cable bacteria are sulfide-oxidising, filamentous bacteria that reduce toxic sulfide levels, suppress methane emissions and drive nutrient and carbon cycling in sediments. Recently, cable bacteria have been found associated with roots of aquatic plants and rice (Oryza sativa). However, the extent to which cable bacteria are associated with aquatic plants in nature remains unexplored. Using newly generated and public 16S rRNA gene sequence datasets combined with fluorescence in situ hybridisation, we investigated the distribution of cable bacteria around the roots of aquatic plants, encompassing seagrass (including seagrass seedlings), rice, freshwater and saltmarsh plants. Diverse cable bacteria were found associated with roots of 16 out of 28 plant species and at 36 out of 55 investigated sites, across four continents. Plant-associated cable bacteria were confirmed across a variety of ecosystems, including marine coastal environments, estuaries, freshwater streams, isolated pristine lakes and intensive agricultural systems. This pattern indicates that this plant-microbe relationship is globally widespread and neither obligate nor species specific. The occurrence of cable bacteria in plant rhizospheres may be of general importance to vegetation vitality, primary productivity, coastal restoration practices and greenhouse gas balance of rice fields and wetlands.

Effect of salinity on cable bacteria species composition and diversity.

Cable bacteria (CB) are Desulfobulbaceae that couple sulphide oxidation to oxygen reduction over centimetre distances by mediating electric currents. Recently, it was suggested that the CB clade is composed of two genera, Ca. Electronema and Ca. Electrothrix, with distinct freshwater and marine habitats respectively. However, only a few studies have reported CB from freshwater sediment, making this distinction uncertain. Here, we report novel data to show that salinity is a controlling factor for the diversity and the species composition within CB populations. CB sampled from a freshwater site (salinity 0.3) grouped into Ca. Electronema and could not grow under brackish conditions (salinity 21), whereas CB from a brackish site (salinity 21) grouped into Ca. Electrothrix and decreased by 93% in activity under freshwater conditions. On a regional scale (Baltic Sea), salinity significantly influenced species richness and composition. However, other environmental factors, such as temperature and quantity and quality of organic matter were also important to explain the observed variation. A global survey of 16S rRNA gene amplicon sequencing revealed that the two genera did not co-occur likely because of competitive exclusion and identified a possible third genus.

Oxygen consumption of individual cable bacteria.

The electric wires of cable bacteria possibly support a unique respiration mode with a few oxygen-reducing cells flaring off electrons, while oxidation of the electron donor and the associated energy conservation and growth is allocated to other cells not exposed to oxygen. Cable bacteria are centimeter-long, multicellular, filamentous Desulfobulbaceae that transport electrons across oxic-anoxic interfaces in aquatic sediments. From observed distortions of the oxic-anoxic interface, we derived oxygen consumption rates of individual cable bacteria and found biomass-specific rates of unheard magnitude in biology. Tightly controlled behavior, possibly involving intercellular electrical signaling, was found to generally keep <10% of individual filaments exposed to oxygen. The results strengthen the hypothesis that cable bacteria indeed have evolved an exceptional way to take the full energetic advantages of aerobic respiration and let >90% of the cells metabolize in the convenient absence of oxidative stress.

Cable bacteria reduce methane emissions from rice-vegetated soils.

Methane is the second most important greenhouse gas after carbon dioxide and approximately 11% of the global anthropogenic methane emissions originate from rice fields. Sulfate amendment is a mitigation strategy to reduce methane emissions from rice fields because sulfate reducers and methanogens compete for the same substrates. Cable bacteria are filamentous bacteria known to increase sulfate levels via electrogenic sulfide oxidation. Here we show that one-time inoculation of rice-vegetated soil pots with cable bacteria increases the sulfate inventory 5-fold, which leads to the reduction of methane emissions by 93%, compared to control pots lacking cable bacteria. Promoting cable bacteria in rice fields by enrichment or sensible management may thus become a strategy to reduce anthropogenic methane emissions.

Electrogenic sulfide oxidation mediated by cable bacteria stimulates sulfate reduction in freshwater sediments.

Cable bacteria are filamentous members of the Desulfobulbaceae family that oxidize sulfide with oxygen or nitrate by transferring electrons over centimeter distances in sediments. Recent studies show that freshwater sediments can support populations of cable bacteria at densities comparable to those found in marine environments. This is surprising since sulfide availability is presumably low in freshwater sediments due to sulfate limitation of sulfate reduction. Here we show that cable bacteria stimulate sulfate reduction in freshwater sediment through promotion of sulfate availability. Comparing experimental freshwater sediments with and without active cable bacteria, we observed a three- to tenfold increase in sulfate concentrations and a 4.5-fold increase in sulfate reduction rates when cable bacteria were present, while abundance and community composition of sulfate-reducing microorganisms (SRM) were unaffected. Correlation and ANCOVA analysis supported the hypothesis that the stimulation of sulfate reduction activity was due to relieve of the kinetic limitations of the SRM community through the elevated sulfate concentrations in sediments with cable bacteria activity. The elevated sulfate concentration was caused by cable bacteria-driven sulfide oxidation, by sulfate production from an indigenous sulfide pool, likely through cable bacteria-mediated dissolution and oxidation of iron sulfides, and by enhanced retention of sulfate, triggered by an electric field generated by the cable bacteria. Cable bacteria in freshwater sediments may thus be an integral component of a cryptic sulfur cycle and provide a mechanism for recycling of the scarce resource sulfate, stimulating sulfate reduction. It is possible that this stimulation has implication for methanogenesis and greenhouse gas emissions.

On the evolution and physiology of cable bacteria.

Cable bacteria of the family Desulfobulbaceae form centimeter-long filaments comprising thousands of cells. They occur worldwide in the surface of aquatic sediments, where they connect sulfide oxidation with oxygen or nitrate reduction via long-distance electron transport. In the absence of pure cultures, we used single-filament genomics and metagenomics to retrieve draft genomes of 3 marine Candidatus Electrothrix and 1 freshwater Ca. Electronema species. These genomes contain >50% unknown genes but still share their core genomic makeup with sulfate-reducing and sulfur-disproportionating Desulfobulbaceae, with few core genes lost and 212 unique genes (from 197 gene families) conserved among cable bacteria. Last common ancestor analysis indicates gene divergence and lateral gene transfer as equally important origins of these unique genes. With support from metaproteomics of a Ca. Electronema enrichment, the genomes suggest that cable bacteria oxidize sulfide by reversing the canonical sulfate reduction pathway and fix CO2 using the Wood-Ljungdahl pathway. Cable bacteria show limited organotrophic potential, may assimilate smaller organic acids and alcohols, fix N2, and synthesize polyphosphates and polyglucose as storage compounds; several of these traits were confirmed by cell-level experimental analyses. We propose a model for electron flow from sulfide to oxygen that involves periplasmic cytochromes, yet-unidentified conductive periplasmic fibers, and periplasmic oxygen reduction. This model proposes that an active cable bacterium gains energy in the anodic, sulfide-oxidizing cells, whereas cells in the oxic zone flare off electrons through intense cathodic oxygen respiration without energy conservation; this peculiar form of multicellularity seems unparalleled in the microbial world.

Transient bottom water oxygenation creates a niche for cable bacteria in long-term anoxic sediments of the Eastern Gotland Basin.

Cable bacteria have been reported in sediments from marine and freshwater locations, but the environmental factors that regulate their growth in natural settings are not well understood. Most prominently, the physiological limit of cable bacteria in terms of oxygen availability remains poorly constrained. In this study, we investigated the presence, activity and diversity of cable bacteria in relation to a natural gradient in bottom water oxygenation in a depth transect of the Eastern Gotland Basin (Baltic Sea). Cable bacteria were identified by FISH at the oxic and transiently oxic sites, but not at the permanently anoxic site. Three species of the candidate genus Electrothrix, i.e. marina, aarhusiensis and communis were found coexisting within one site. The highest filament density (33 m cm-2 ) was associated with a 6.3 mm wide zone depleted in both oxygen and free sulphide, and the presence of an electric field resulting from the electrogenic sulphur oxidizing metabolism of cable bacteria. However, the measured filament densities and metabolic activities remained low overall, suggesting a limited impact of cable bacteria at the basin level. The observed bottom water oxygen levels (< 5 µM) are the lowest so far reported for cable bacteria, thus expanding their known environmental distribution.

Concurrent Methane Production and Oxidation in Surface Sediment from Aarhus Bay, Denmark.

Marine surface sediments, which are replete with sulfate, are typically considered to be devoid of endogenous methanogenesis. Yet, methanogenic archaea are present in those sediments, suggesting a potential for methanogenesis. We used an isotope dilution method based on sediment bag incubation and spiking with 13C-CH4 to quantify CH4 turnover rates in sediment from Aarhus Bay, Denmark. In two independent experiments, highest CH4 production and oxidation rates (>200 pmol cm-3 d-1) were found in the top 0-2 cm, below which rates dropped below 100 pmol cm-3 d-1 in all other segments down to 16 cm. This drop in overall methane turnover with depth was accompanied by decreasing rates of organic matter mineralization with depth. Molecular analyses based on quantitative PCR and MiSeq sequencing of archaeal 16S rRNA genes showed that the abundance of methanogenic archaea also peaked in the top 0-2 cm segment. Based on the community profiling, hydrogenotrophic and methylotrophic methanogens dominated among the methanogenic archaea in general, suggesting that methanogenesis in surface sediment could be driven by both CO2 reduction and fermentation of methylated compounds. Our results show the existence of elevated methanogenic activity and a dynamic recycling of CH4 at low concentration in sulfate-rich marine surface sediment. Considering the common environmental conditions found in other coastal systems, we speculate that such a cryptic methane cycling can be ubiquitous.

Sinks and Sources of Intracellular Nitrate in Gromiids.

A substantial nitrate pool is stored within living cells in various benthic marine environments. The fate of this bioavailable nitrogen differs according to the organisms managing the intracellular nitrate (ICN). While some light has been shed on the nitrate carried by diatoms and foraminiferans, no study has so far followed the nitrate kept by gromiids. Gromiids are large protists and their ICN concentration can exceed 1000x the ambient nitrate concentration. In the present study we investigated gromiids from diverse habitats and showed that they contained nitrate at concentrations ranging from 1 to 370 mM. We used 15N tracer techniques to investigate the source of this ICN, and found that it originated both from active nitrate uptake from the environment and from intracellular production, most likely through bacterial nitrification. Microsensor measurements showed that part of the ICN was denitrified to N2 when gromiids were exposed to anoxia. Denitrification seemed to be mediated by endobiotic bacteria because antibiotics inhibited denitrification activity. The active uptake of nitrate suggests that ICN plays a role in gromiid physiology and is not merely a consequence of the gromiid hosting a diverse bacterial community. Measurements of aerobic respiration rates and modeling of oxygen consumption by individual gromiid cells suggested that gromiids may occasionally turn anoxic by their own respiration activity and thus need strategies for coping with this self-inflicted anoxia.

Electrogenic sulfur oxidation in a northern saltmarsh (St. Lawrence Estuary, Canada).

Measurements of porewater O2, pH, and H2S microprofiles in intact sediment cores collected in a northern saltmarsh in the St. Lawrence Estuary (Quebec, Canada) revealed the occurrence of electrogenic sulfur oxidation (e-SOx) by filamentous "cable" bacteria in submerged marsh pond sediments in the high marsh. In summer, the geochemical fingerprint of e-SOx was apparent in intact cores, while in fall, cable bacteria were detected by fluorescence in situ hybridization and the characteristic geochemical signature of e-SOx was observed only upon prolonged incubation. In exposed, unvegetated creek bank sediments sampled in the low marsh in summer, cable bacteria developed only in repacked cores of sieved (500 µm), homogenized sediments. These results suggest that e-SOx is suppressed by the activity of macrofauna in exposed, unvegetated marsh sediments. A reduced abundance of benthic invertebrates may promote e-SOx development in marsh ponds, which are dominant features of subarctic saltmarshes as in the St. Lawrence Estuary.

Cable Bacteria in Freshwater Sediments.

In marine sediments cathodic oxygen reduction at the sediment surface can be coupled to anodic sulfide oxidation in deeper anoxic layers through electrical currents mediated by filamentous, multicellular bacteria of the Desulfobulbaceae family, the so-called cable bacteria. Until now, cable bacteria have only been reported from marine environments. In this study, we demonstrate that cable bacteria also occur in freshwater sediments. In a first step, homogenized sediment collected from the freshwater stream Giber Å, Denmark, was incubated in the laboratory. After 2 weeks, pH signatures and electric fields indicated electron transfer between vertically separated anodic and cathodic half-reactions. Fluorescence in situ hybridization revealed the presence of Desulfobulbaceae filaments. In addition, in situ measurements of oxygen, pH, and electric potential distributions in the waterlogged banks of Giber Å demonstrated the presence of distant electric redox coupling in naturally occurring freshwater sediment. At the same site, filamentous Desulfobulbaceae with cable bacterium morphology were found to be present. Their 16S rRNA gene sequence placed them as a distinct sister group to the known marine cable bacteria, with the genus Desulfobulbus as the closest cultured lineage. The results of the present study indicate that electric currents mediated by cable bacteria could be important for the biogeochemistry in many more environments than anticipated thus far and suggest a common evolutionary origin of the cable phenotype within Desulfobulbaceae with subsequent diversification into a freshwater and a marine lineage.

Rethinking sediment biogeochemistry after the discovery of electric currents.

The discovery of electric currents in marine sediments arose from a simple observation that conventional biogeochemistry could not explain: Sulfide oxidation in one place is closely coupled to oxygen reduction in another place, centimeters away. After experiments demonstrated that this resulted from electric coupling, the conductors were found to be long, multicellular, filamentous bacteria, now known as cable bacteria. The spatial separation of oxidation and reduction processes by these bacteria represents a shortcut in the conventional cascade of redox processes and may drive most of the oxygen consumption. In addition, it implies a separation of strong proton generators and consumers and the formation of measurable electric fields, which have several effects on mineral development and ion migration. This article reviews the work on electric currents and cable bacteria published through April 2014, with an emphasis on general trends, thought-provoking consequences, and new questions to address.

Nitrate Storage and Dissimilatory Nitrate Reduction by Eukaryotic Microbes.

The microbial nitrogen cycle is one of the most complex and environmentally important element cycles on Earth and has long been thought to be mediated exclusively by prokaryotic microbes. Rather recently, it was discovered that certain eukaryotic microbes are able to store nitrate intracellularly and use it for dissimilatory nitrate reduction in the absence of oxygen. The paradigm shift that this entailed is ecologically significant because the eukaryotes in question comprise global players like diatoms, foraminifers, and fungi. This review article provides an unprecedented overview of nitrate storage and dissimilatory nitrate reduction by diverse marine eukaryotes placed into an eco-physiological context. The advantage of intracellular nitrate storage for anaerobic energy conservation in oxygen-depleted habitats is explained and the life style enabled by this metabolic trait is described. A first compilation of intracellular nitrate inventories in various marine sediments is presented, indicating that intracellular nitrate pools vastly exceed porewater nitrate pools. The relative contribution by foraminifers to total sedimentary denitrification is estimated for different marine settings, suggesting that eukaryotes may rival prokaryotes in terms of dissimilatory nitrate reduction. Finally, this review article sketches some evolutionary perspectives of eukaryotic nitrate metabolism and identifies open questions that need to be addressed in future investigations.

Electric coupling between distant nitrate reduction and sulfide oxidation in marine sediment.

Filamentous bacteria of the Desulfobulbaceae family can conduct electrons over centimeter-long distances thereby coupling oxygen reduction at the surface of marine sediment to sulfide oxidation in deeper anoxic layers. The ability of these cable bacteria to use alternative electron acceptors is currently unknown. Here we show that these organisms can use also nitrate or nitrite as an electron acceptor thereby coupling the reduction of nitrate to distant oxidation of sulfide. Sulfidic marine sediment was incubated with overlying nitrate-amended anoxic seawater. Within 2 months, electric coupling of spatially segregated nitrate reduction and sulfide oxidation was evident from: (1) the formation of a 4-6-mm-deep zone separating sulfide oxidation from the associated nitrate reduction, and (2) the presence of pH signatures consistent with proton consumption by cathodic nitrate reduction, and proton production by anodic sulfide oxidation. Filamentous Desulfobulbaceae with the longitudinal structures characteristic of cable bacteria were detected in anoxic, nitrate-amended incubations but not in anoxic, nitrate-free controls. Nitrate reduction by cable bacteria using long-distance electron transport to get privileged access to distant electron donors is a hitherto unknown mechanism in nitrogen and sulfur transformations, and the quantitative importance for elements cycling remains to be addressed.

Succession of cable bacteria and electric currents in marine sediment.

Filamentous Desulfobulbaceae have been reported to conduct electrons over centimetre-long distances, thereby coupling oxygen reduction at the surface of marine sediment to sulphide oxidation in sub-surface layers. To understand how these 'cable bacteria' establish and sustain electric conductivity, we followed a population for 53 days after exposing sulphidic sediment with initially no detectable filaments to oxygen. After 10 days, cable bacteria and electric currents were established throughout the top 15 mm of the sediment, and after 21 days the filament density peaked with a total length of 2 km cm(-2). Cells elongated and divided at all depths with doubling times over the first 10 days of <20 h. Active, oriented movement must have occurred to explain the separation of O2 and H2S by 15 mm. Filament diameters varied from 0.4-1.7 µm, with a general increase over time and depth, and yet they shared 16S rRNA sequence identity of >98%. Comparison of the increase in biovolume and electric current density suggested high cellular growth efficiency. While the vertical expansion of filaments continued over time and reached 30 mm, the electric current density and biomass declined after 13 and 21 days, respectively. This might reflect a breakdown of short filaments as their solid sulphide sources became depleted in the top layers of the anoxic zone. In conclusion, cable bacteria combine rapid and efficient growth with oriented movement to establish and exploit the spatially separated half-reactions of sulphide oxidation and oxygen consumption.

Filamentous bacteria transport electrons over centimetre distances.

Oxygen consumption in marine sediments is often coupled to the oxidation of sulphide generated by degradation of organic matter in deeper, oxygen-free layers. Geochemical observations have shown that this coupling can be mediated by electric currents carried by unidentified electron transporters across centimetre-wide zones. Here we present evidence that the native conductors are long, filamentous bacteria. They abounded in sediment zones with electric currents and along their length they contained strings with distinct properties in accordance with a function as electron transporters. Living, electrical cables add a new dimension to the understanding of interactions in nature and may find use in technology development.

Anaerobic ammonia oxidation in a fertilized paddy soil.

Evidence for anaerobic ammonium oxidation in a paddy field was obtained in Southern China using an isotope-pairing technique, quantitative PCR assays and 16S rRNA gene clone libraries, along with nutrient profiles of soil cores. A paddy field with a high load of slurry manure as fertilizer was selected for this study and was shown to contain a high amount of ammonium (6.2-178.8 mg kg(-1)). The anaerobic oxidation of ammonium (anammox) rates in this paddy soil ranged between 0.5 and 2.9 nmol N per gram of soil per hour in different depths of the soil core, and the specific cellular anammox activity observed in batch tests ranged from 2.9 to 21 fmol per cell per day. Anammox contributed 4-37% to soil N2 production, the remainder being due to denitrification. The 16S rRNA gene sequences of surface soil were closely related to the anammox bacteria 'Kuenenia', 'Anammoxoglobus' and 'Jettenia'. Most of the anammox 16S rRNA genes retrieved from the deeper soil were affiliated to 'Brocadia'. The retrieval of mainly bacterial amoA sequences in the upper part of the paddy soil indicated that nitrifying bacteria may be the major source of nitrite for anammox bacteria in the cultivated horizon. In the deeper oxygen-limited parts, only archaeal amoA sequences were found, indicating that archaea may produce nitrite in this part of the soil. It is estimated that a total loss of 76 g N m(-2) per year is linked to anammox in the paddy field.