Persistent activity of aerobic methane-oxidizing bacteria in anoxic lake waters due to metabolic versatility.

Lacustrine methane emissions are strongly mitigated by aerobic methane-oxidizing bacteria (MOB) that are typically most active at the oxic-anoxic interface. Although oxygen is required by the MOB for the first step of methane oxidation, their occurrence in anoxic lake waters has raised the possibility that they are capable of oxidizing methane further anaerobically. Here, we investigate the activity and growth of MOB in Lake Zug, a permanently stratified freshwater lake. The rates of anaerobic methane oxidation in the anoxic hypolimnion reached up to 0.2 µM d-1. Single-cell nanoSIMS measurements, together with metagenomic and metatranscriptomic analyses, linked the measured rates to MOB of the order Methylococcales. Interestingly, their methane assimilation activity was similar under hypoxic and anoxic conditions. Our data suggest that these MOB use fermentation-based methanotrophy as well as denitrification under anoxic conditions, thus offering an explanation for their widespread presence in anoxic habitats such as stratified water columns. Thus, the methane sink capacity of anoxic basins may have been underestimated by not accounting for the anaerobic MOB activity.

Rhizobia-diatom symbiosis fixes missing nitrogen in the ocean.

Nitrogen (N2) fixation in oligotrophic surface waters is the main source of new nitrogen to the ocean1 and has a key role in fuelling the biological carbon pump2. Oceanic N2 fixation has been attributed almost exclusively to cyanobacteria, even though genes encoding nitrogenase, the enzyme that fixes N2 into ammonia, are widespread among marine bacteria and archaea3-5. Little is known about these non-cyanobacterial N2 fixers, and direct proof that they can fix nitrogen in the ocean has so far been lacking. Here we report the discovery of a non-cyanobacterial N2-fixing symbiont, 'Candidatus Tectiglobus diatomicola', which provides its diatom host with fixed nitrogen in return for photosynthetic carbon. The N2-fixing symbiont belongs to the order Rhizobiales and its association with a unicellular diatom expands the known hosts for this order beyond the well-known N2-fixing rhizobia-legume symbioses on land6. Our results show that the rhizobia-diatom symbioses can contribute as much fixed nitrogen as can cyanobacterial N2 fixers in the tropical North Atlantic, and that they might be responsible for N2 fixation in the vast regions of the ocean in which cyanobacteria are too rare to account for the measured rates.

Methylphosphonate-driven methane formation and its link to primary production in the oligotrophic North Atlantic.

Methylphosphonate is an organic phosphorus compound used by microorganisms when phosphate, a key nutrient limiting growth in most marine surface waters, becomes unavailable. Microbial methylphosphonate use can result in the formation of methane, a potent greenhouse gas, in oxic waters where methane production is traditionally unexpected. The extent and controlling factors of such aerobic methane formation remain underexplored. Here, we show high potential net rates of methylphosphonate-driven methane formation (median 0.4 nmol methane L-1 d-1) in the upper water column of the western tropical North Atlantic. The rates are repressed but still quantifiable in the presence of in-situ or added phosphate, suggesting that some methylphosphonate-driven methane formation persists in phosphate-replete waters. The genetic potential for methylphosphonate utilisation is present in and transcribed by key photo- and heterotrophic microbial taxa, such as Pelagibacterales, SAR116, and Trichodesmium. While the large cyanobacterial nitrogen-fixers dominate in the surface layer, phosphonate utilisation by Alphaproteobacteria appears to become more important in deeper depths. We estimate that at our study site, a substantial part (median 11%) of the measured surface carbon fixation can be sustained by phosphorus liberated from phosphonate utilisation, highlighting the ecological importance of phosphonates in the carbon cycle of the oligotrophic ocean.

Simultaneous visualization of flow fields and oxygen concentrations to unravel transport and metabolic processes in biological systems.

From individual cells to whole organisms, O2 transport unfolds across micrometer- to millimeter-length scales and can change within milliseconds in response to fluid flows and organismal behavior. The spatiotemporal complexity of these processes makes the accurate assessment of O2 dynamics via currently available methods difficult or unreliable. Here, we present "sensPIV," a method to simultaneously measure O2 concentrations and flow fields. By tracking O2-sensitive microparticles in flow using imaging technologies that allow for instantaneous referencing, we measured O2 transport within (1) microfluidic devices, (2) sinking model aggregates, and (3) complex colony-forming corals. Through the use of sensPIV, we find that corals use ciliary movement to link zones of photosynthetic O2 production to zones of O2 consumption. SensPIV can potentially be extendable to study flow-organism interactions across many life-science and engineering applications.

Quantification of archaea-driven freshwater nitrification from single cell to ecosystem levels.

Deep oligotrophic lakes sustain large populations of the class Nitrososphaeria (Thaumarchaeota) in their hypolimnion. They are thought to be the key ammonia oxidizers in this habitat, but their impact on N-cycling in lakes has rarely been quantified. We followed this archaeal population in one of Europe's largest lakes, Lake Constance, for two consecutive years using metagenomics and metatranscriptomics combined with stable isotope-based activity measurements. An abundant (8-39% of picoplankton) and transcriptionally active archaeal ecotype dominated the nitrifying community. It represented a freshwater-specific species present in major inland water bodies, for which we propose the name "Candidatus Nitrosopumilus limneticus". Its biomass corresponded to 12% of carbon stored in phytoplankton over the year´s cycle. Ca. N. limneticus populations incorporated significantly more ammonium than most other microorganisms in the hypolimnion and were driving potential ammonia oxidation rates of 6.0 ± 0.9 nmol l‒1 d‒1, corresponding to potential cell-specific rates of 0.21 ± 0.11 fmol cell-1 d-1. At the ecosystem level, this translates to a maximum capacity of archaea-driven nitrification of 1.76 × 109 g N-ammonia per year or 11% of N-biomass produced annually by phytoplankton. We show that ammonia-oxidizing archaea play an equally important role in the nitrogen cycle of deep oligotrophic lakes as their counterparts in marine ecosystems.

Diverse methylotrophic methanogenic archaea cause high methane emissions from seagrass meadows.

Marine coastlines colonized by seagrasses are a net source of methane to the atmosphere. However, methane emissions from these environments are still poorly constrained, and the underlying processes and responsible microorganisms remain largely unknown. Here, we investigated methane turnover in seagrass meadows of Posidonia oceanica in the Mediterranean Sea. The underlying sediments exhibited median net fluxes of methane into the water column of ca. 106 µmol CH4 ⋅ m-2 ⋅ d-1 Our data show that this methane production was sustained by methylated compounds produced by the plant, rather than by fermentation of buried organic carbon. Interestingly, methane production was maintained long after the living plant died off, likely due to the persistence of methylated compounds, such as choline, betaines, and dimethylsulfoniopropionate, in detached plant leaves and rhizomes. We recovered multiple mcrA gene sequences, encoding for methyl-coenzyme M reductase (Mcr), the key methanogenic enzyme, from the seagrass sediments. Most retrieved mcrA gene sequences were affiliated with a clade of divergent Mcr and belonged to the uncultured Candidatus Helarchaeota of the Asgard superphylum, suggesting a possible involvement of these divergent Mcr in methane metabolism. Taken together, our findings identify the mechanisms controlling methane emissions from these important blue carbon ecosystems.

How low can they go? Aerobic respiration by microorganisms under apparent anoxia.

Oxygen (O2) is the ultimate oxidant on Earth and its respiration confers such an energetic advantage that microorganisms have evolved the capacity to scavenge O2 down to nanomolar concentrations. The respiration of O2 at extremely low levels is proving to be common to diverse microbial taxa, including organisms formerly considered strict anaerobes. Motivated by recent advances in O2 sensing and DNA/RNA sequencing technologies, we performed a systematic review of environmental metatranscriptomes revealing that microbial respiration of O2 at nanomolar concentrations is ubiquitous and drives microbial activity in seemingly anoxic aquatic habitats. These habitats were key to the early evolution of life and are projected to become more prevalent in the near future due to anthropogenic-driven environmental change. Here, we summarize our current understanding of aerobic microbial respiration under apparent anoxia, including novel processes, their underlying biochemical pathways, the involved microorganisms, and their environmental importance and evolutionary origin.

Niche partitioning by photosynthetic plankton as a driver of CO2-fixation across the oligotrophic South Pacific Subtropical Ocean.

Oligotrophic ocean gyre ecosystems may be expanding due to rising global temperatures [1-5]. Models predicting carbon flow through these changing ecosystems require accurate descriptions of phytoplankton communities and their metabolic activities [6]. We therefore measured distributions and activities of cyanobacteria and small photosynthetic eukaryotes throughout the euphotic zone on a zonal transect through the South Pacific Ocean, focusing on the ultraoligotrophic waters of the South Pacific Gyre (SPG). Bulk rates of CO2 fixation were low (0.1 µmol C l-1 d-1) but pervasive throughout both the surface mixed-layer (upper 150 m), as well as the deep chlorophyll a maximum of the core SPG. Chloroplast 16S rRNA metabarcoding, and single-cell 13CO2 uptake experiments demonstrated niche differentiation among the small eukaryotes and picocyanobacteria. Prochlorococcus abundances, activity, and growth were more closely associated with the rims of the gyre. Small, fast-growing, photosynthetic eukaryotes, likely related to the Pelagophyceae, characterized the deep chlorophyll a maximum. In contrast, a slower growing population of photosynthetic eukaryotes, likely comprised of Dictyochophyceae and Chrysophyceae, dominated the mixed layer that contributed 65-88% of the areal CO2 fixation within the core SPG. Small photosynthetic eukaryotes may thus play an underappreciated role in CO2 fixation in the surface mixed-layer waters of ultraoligotrophic ecosystems.

The rate and fate of N2 and C fixation by marine diatom-diazotroph symbioses.

N2 fixation constitutes an important new nitrogen source in the open sea. One group of filamentous N2 fixing cyanobacteria (Richelia intracellularis, hereafter Richelia) form symbiosis with a few genera of diatoms. High rates of N2 fixation and carbon (C) fixation have been measured in the presence of diatom-Richelia symbioses. However, it is unknown how partners coordinate C fixation and how the symbiont sustains high rates of N2 fixation. Here, both the N2 and C fixation in wild diatom-Richelia populations are reported. Inhibitor experiments designed to inhibit host photosynthesis, resulted in lower estimated growth and depressed C and N2 fixation, suggesting that despite the symbionts ability to fix their own C, they must still rely on their respective hosts for C. Single cell analysis indicated that up to 22% of assimilated C in the symbiont is derived from the host, whereas 78-91% of the host N is supplied from their symbionts. A size-dependent relationship is identified where larger cells have higher N2 and C fixation, and only N2 fixation was light dependent. Using the single cell measures, the N-rich phycosphere surrounding these symbioses was estimated and contributes directly and rapidly to the surface ocean rather than the mesopelagic, even at high estimated sinking velocities (<10 m d-1). Several eco-physiological parameters necessary for incorporating symbiotic N2 fixing populations into larger basin scale biogeochemical models (i.e., N and C cycles) are provided.

Terrestrial-type nitrogen-fixing symbiosis between seagrass and a marine bacterium.

Symbiotic N2-fixing microorganisms have a crucial role in the assimilation of nitrogen by eukaryotes in nitrogen-limited environments1-3. Particularly among land plants, N2-fixing symbionts occur in a variety of distantly related plant lineages and often involve an intimate association between host and symbiont2,4. Descriptions of such intimate symbioses are lacking for seagrasses, which evolved around 100 million years ago from terrestrial flowering plants that migrated back to the sea5. Here we describe an N2-fixing symbiont, 'Candidatus Celerinatantimonas neptuna', that lives inside seagrass root tissue, where it provides ammonia and amino acids to its host in exchange for sugars. As such, this symbiosis is reminiscent of terrestrial N2-fixing plant symbioses. The symbiosis between Ca. C. neptuna and its host Posidonia oceanica enables highly productive seagrass meadows to thrive in the nitrogen-limited Mediterranean Sea. Relatives of Ca. C. neptuna occur worldwide in coastal ecosystems, in which they may form similar symbioses with other seagrasses and saltmarsh plants. Just like N2-fixing microorganisms might have aided the colonization of nitrogen-poor soils by early land plants6, the ancestors of Ca. C. neptuna and its relatives probably enabled flowering plants to invade nitrogen-poor marine habitats, where they formed extremely efficient blue carbon ecosystems7.

Purple sulfur bacteria fix N2 via molybdenum-nitrogenase in a low molybdenum Proterozoic ocean analogue.

Biological N2 fixation was key to the expansion of life on early Earth. The N2-fixing microorganisms and the nitrogenase type used in the Proterozoic are unknown, although it has been proposed that the canonical molybdenum-nitrogenase was not used due to low molybdenum availability. We investigate N2 fixation in Lake Cadagno, an analogue system to the sulfidic Proterozoic continental margins, using a combination of biogeochemical, molecular and single cell techniques. In Lake Cadagno, purple sulfur bacteria (PSB) are responsible for high N2 fixation rates, to our knowledge providing the first direct evidence for PSB in situ N2 fixation. Surprisingly, no alternative nitrogenases are detectable, and N2 fixation is exclusively catalyzed by molybdenum-nitrogenase. Our results show that molybdenum-nitrogenase is functional at low molybdenum conditions in situ and that in contrast to previous beliefs, PSB may have driven N2 fixation in the Proterozoic ocean.

Small sinking particles control anammox rates in the Peruvian oxygen minimum zone.

Anaerobic oxidation of ammonium (anammox) in oxygen minimum zones (OMZs) is a major pathway of oceanic nitrogen loss. Ammonium released from sinking particles has been suggested to fuel this process. During cruises to the Peruvian OMZ in April-June 2017 we found that anammox rates are strongly correlated with the volume of small particles (128-512 µm), even though anammox bacteria were not directly associated with particles. This suggests that the relationship between anammox rates and particles is related to the ammonium released from particles by remineralization. To investigate this, ammonium release from particles was modelled and theoretical encounters of free-living anammox bacteria with ammonium in the particle boundary layer were calculated. These results indicated that small sinking particles could be responsible for ~75% of ammonium release in anoxic waters and that free-living anammox bacteria frequently encounter ammonium in the vicinity of smaller particles. This indicates a so far underestimated role of abundant, slow-sinking small particles in controlling oceanic nutrient budgets, and furthermore implies that observations of the volume of small particles could be used to estimate N-loss across large areas.

Anaerobic endosymbiont generates energy for ciliate host by denitrification.

Mitochondria are specialized eukaryotic organelles that have a dedicated function in oxygen respiration and energy production. They evolved about 2 billion years ago from a free-living bacterial ancestor (probably an alphaproteobacterium), in a process known as endosymbiosis1,2. Many unicellular eukaryotes have since adapted to life in anoxic habitats and their mitochondria have undergone further reductive evolution3. As a result, obligate anaerobic eukaryotes with mitochondrial remnants derive their energy mostly from fermentation4. Here we describe 'Candidatus Azoamicus ciliaticola', which is an obligate endosymbiont of an anaerobic ciliate and has a dedicated role in respiration and providing energy for its eukaryotic host. 'Candidatus A. ciliaticola' contains a highly reduced 0.29-Mb genome that encodes core genes for central information processing, the electron transport chain, a truncated tricarboxylic acid cycle, ATP generation and iron-sulfur cluster biosynthesis. The genome encodes a respiratory denitrification pathway instead of aerobic terminal oxidases, which enables its host to breathe nitrate instead of oxygen. 'Candidatus A. ciliaticola' and its ciliate host represent an example of a symbiosis that is based on the transfer of energy in the form of ATP, rather than nutrition. This discovery raises the possibility that eukaryotes with mitochondrial remnants may secondarily acquire energy-providing endosymbionts to complement or replace functions of their mitochondria.

Assigning Function to Phylogeny: FISH-nanoSIMS.

Catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) is an imaging method used to identify microorganisms in environmental samples based on their phylogeny. CARD-FISH can be combined with nano-scale secondary ion mass spectrometry (nanoSIMS) to directly link the cell identity to their activity, measured as the incorporation of stable isotopes into hybridized cells after stable isotope probing. In environmental microbiology, a combination of these methods has been used to determine the identity and growth of uncultured microorganisms, and to explore the factors controlling their activity. Additionally, FISH-nanoSIMS has been widely used to directly visualize microbial interactions in situ. Here, we describe a step-by-step protocol for a combination of CARD-FISH, laser marking, and nanoSIMS analysis on samples from aquatic environments.

Rapid microbial diversification of dissolved organic matter in oceanic surface waters leads to carbon sequestration.

The pool of dissolved organic matter (DOM) in the deep ocean represents one of the largest carbon sinks on the planet. In recent years, studies have shown that most of this pool is recalcitrant, because individual compounds are present at low concentrations and because certain compounds seem resistant to microbial degradation. The formation of the diverse and recalcitrant deep ocean DOM pool has been attributed to repeated and successive processing of DOM by microorganisms over time scales of weeks to years. Little is known however, about the transformation and cycling that labile DOM undergoes in the first hours upon its release from phytoplankton. Here we provide direct experimental evidence showing that within hours of labile DOM release, its breakdown and recombination with ambient DOM leads to the formation of a diverse array of new molecules in oligotrophic North Atlantic surface waters. Furthermore, our results reveal a preferential breakdown of N and P containing molecules versus those containing only carbon. Hence, we show the preferential breakdown and molecular diversification are the crucial first steps in the eventual formation of carbon rich DOM that is resistant to microbial remineralization.

An intracellular silver deposition method for targeted detection and chemical analysis of uncultured microorganisms.

The vast majority of environmental bacteria remain uncultured, despite two centuries of effort in cultivating microorganisms. Our knowledge of their physiology and metabolic activity depends to a large extent on methods capable of analyzing single cells. Bacterial identification is a key step required by all currently used single-cell imaging techniques and is typically performed by means of fluorescent labeling. However, fluorescent cells cannot be visualized by ion- and electron microscopy and thus only correlative, indirect, cell identification is possible. Here we present a new method of bacterial identification by in situ hybridization coupled to the deposition of elemental silver nanoparticles (silver-DISH). We show that hybridized cells containing silver can be directly visualized by light microscopy, scanning electron microscopy, energy dispersive X-ray spectroscopy, secondary ion mass spectrometry (nanoSIMS), and confocal Raman micro-spectroscopy. Silver-DISH did not alter the isotopic (13C) and elemental composition of stable-isotope probed cells more than other available hybridization methods, making silver-DISH suitable for broad applications in stable-isotope labeling studies. Additionally, we demonstrate that silver-DISH can induce a surface-enhanced Raman scattering (SERS) effect, amplifying the Raman signal of biomolecules inside bacterial cells. This makes silver-DISH the only currently available method that is capable of delivering a SERS-active substrate inside specifically targeted microbial cells.

The effect of sediment grain properties and porewater flow on microbial abundance and respiration in permeable sediments.

Sandy sediments cover 50-60% of the continental shelves and are highly efficient bioreactors in which organic carbon is remineralized and inorganic nitrogen is reduced to N2. As such they seem to play an important role, buffering the open ocean from anthropogenic nitrogen inputs and likely remineralizing the vast amounts of organic matter formed in the highly productive surface waters. To date however, little is known about the interrelation between porewater transport, grain properties and microbial colonization and the consequences for remineralization rates in sandy sediments. To constrain the effect of theses factors on remineralization in silicate sands, we incubated North Sea sediments in flow-through reactors after separating into five different grain size fractions. Bulk sediment and sediment grain properties were measured along with microbial colonization and cell abundances, oxygen consumption and denitrification rates. Volumetric oxygen consumption ranged from 14 to 77 µmol O2 l-1 h-1 while nitrogen-loss via denitrification was between 3.7 and 8.4 µmol N l-1 h-1. Oxygen consumption and denitrification rates were linearly correlated to the microbial cell abundances, which ranged from 2.9 to 5.4·108 cells cm-3. We found, that cell abundance and consumption rates in sandy sediments are influenced (i) by the surface area available for microbial colonization and (ii) by the exposure of these surfaces to the solute-supplying porewater flow. While protective structures such as cracks and depressions promote microbial colonization, the oxygen demand is only met by good ventilation of these structures, which is supported by a high sphericity of the grains. Based on our results, spherical sand grains with small depressions, i.e. golf ball like structures, provide the optimal supporting mineral structure for microorganisms on continental shelves.

Single cell analyses reveal contrasting life strategies of the two main nitrifiers in the ocean.

Nitrification, the oxidation of ammonia via nitrite to nitrate, is a key process in marine nitrogen (N) cycling. Although oceanic ammonia and nitrite oxidation are balanced, ammonia-oxidizing archaea (AOA) vastly outnumber the main nitrite oxidizers, the bacterial Nitrospinae. The ecophysiological reasons for this discrepancy in abundance are unclear. Here, we compare substrate utilization and growth of Nitrospinae to AOA in the Gulf of Mexico. Based on our results, more than half of the Nitrospinae cellular N-demand is met by the organic-N compounds urea and cyanate, while AOA mainly assimilate ammonium. Nitrospinae have, under in situ conditions, around four-times higher biomass yield and five-times higher growth rates than AOA, despite their ten-fold lower abundance. Our combined results indicate that differences in mortality between Nitrospinae and AOA, rather than thermodynamics, biomass yield and cell size, determine the abundances of these main marine nitrifiers. Furthermore, there is no need to invoke yet undiscovered, abundant nitrite oxidizers to explain nitrification rates in the ocean.

Anaerobic ammonium oxidation is a major N-sink in aquifer systems around the world.

Global-scale N-oxide contamination of groundwater within aquifers occurs due to the widespread use of N-bearing fertilizers and chemicals, threatening both human and environmental health. However, the conversion of these pollutants in active nitrogen (N) cycling processes in the subsurface biosphere still remains unclear. This study investigates the global occurrence of anaerobic ammonium oxidation (anammox) in aquifers, where anammox was found to be turned on and off between saturated and unsaturated soil horizons, and contributed 36.8-79.5% to N loss in saturated soil horizons, the remainder being due to denitrification which has traditionally been considered the main pathway for removal of N-pollutants from aquifers. Although anammox activity was undetectable in the unsaturated soil horizons, it could potentially be activated by contact with ascending groundwater. High-throughput pyrosequencing analysis identified Candidatus Brocadia anammoxidans as being the most abundant anammox bacterium in the saturated soils investigated. However, the anammox bacterial abundance was determined by the relative richness of Candidatus Jettenia asiatica. Isotopic pairing experiments revealed that coupling anammox with ammonium oxidation and respiratory ammonification enabled the formation of a revised N cycle in aquifer systems, in which respiratory ammonification acted as an important coordinator. Anammox can therefore contribute substantially to aquifer N cycling and its role in remediation of aquifers contaminated with N-oxides may be of global importance.