Resolution of Conflicting Signals at the Single-Cell Level in the Regulation of Cyanobacterial Photosynthesis and Nitrogen Fixation

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N-2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N-2 fixation in the dark and photosynthesis in the light. On the population level, this separation is not always complete, since the two processes can overlap during transitions from dark to light. How do single cells avoid inactivation of nitrogenase during these periods? One possibility is that phenotypic heterogeneity in populations leads to segregation of the two processes. Here, we measured N-2 fixation and photosynthesis of individual cells using nanometer-scale secondary ion mass spectrometry (nanoSIMS) to assess both processes in a culture of the unicellular, diazotrophic cyanobacterium Crocosphaera watsonii during a dark-light and a continuous light phase. We compared single-cell rates with bulk rates and gene expression profiles. During the regular dark and light phases, C. watsonii exhibited the temporal segregation of N-2 fixation and photosynthesis commonly observed. However, N-2 fixation and photosynthesis were concurrently measurable at the population level during the subjective dark phase in which cells were kept in the light rather than returned to the expected dark phase. At the single-cell level, though, cells discriminated against either one of the two processes. Cells that showed high levels of photosynthesis had low nitrogen fixing activities, and vice versa. These results suggest that, under ambiguous environmental signals, single cells discriminate against either photosynthesis or nitrogen fixation, and thereby might reduce costs associated with running incompatible processes in the same cell

Extensive nitrogen loss from permeable sediments off North-West Africa

The upwelling area off North-West Africa is characterized by high export production, high nitrate and low oxygen concentration in bottom waters. The underlying sediment consists of sands that cover most of the continental shelf. Due to their permeability sands allow for fast advective pore water transport and can exhibit high rates of nitrogen (N) loss via denitrification as reported for anthropogenically eutrophied regions. However, N loss from sands underlying naturally eutrophied waters is not well studied, and in particular, N loss from the North-West African shelf is poorly constrained. During two research cruises in April/May 2010/2011, sediment was sampled along the North-West African shelf and volumetric denitrification rates were measured in sediment layers down to 8 cm depth using slurry incubations with 15N-labeled nitrate. Areal N loss was calculated by integrating volumetric rates down to the nitrate penetration depth derived from pore water profiles. Areal N loss was neither correlated with water depth nor with bottom water concentrations of nitrate and oxygen but was strongly dependent on sediment grain size and permeability. The derived empirical relation between benthic N loss and grains size suggests that pore water advection is an important regulating parameter for benthic denitrification in sands and further allowed extrapolating rates to an area of 53,000 km2 using detailed sediment maps. Denitrification from this region amounts to 995 kt yr-1 (average 3.6 mmol m-2 d-1) which is 4 times higher than previous estimates based on diffusive pore water transport. Sandy sediments cover 50-60% of the continental shelf and thus may contribute significantly to the global benthic N loss

Aquatic Respiration Rate Measurements at Low Oxygen Concentrations

Despite its huge ecological importance, microbial oxygen respiration in pelagic waters is little studied, primarily due to methodological difficulties. Respiration measurements are challenging because of the required high resolution of oxygen concentration measurements. Recent improvements in oxygen sensing techniques bear great potential to overcome these limitations. Here we compare 3 different methods to measure oxygen consumption rates at low oxygen concentrations, utilizing amperometric Clark type sensors (STOX), optical sensors (optodes), and mass spectrometry in combination with 18-18O2 labeling. Oxygen concentrations and consumption rates agreed well between the different methods when applied in the same experimental setting. Oxygen consumption rates between 30 and 400 nmol L−1 h−1 were measured with high precision and relative standard errors of less than 3%. Rate detection limits in the range of 1 nmol L−1 h−1 were suitable for rate determinations in open ocean water and were lowest at the lowest applied O2 concentration

Oxygen Sensitivity of Anammox and Coupled N-Cycle Processes in Oxygen Minimum Zones

Nutrient measurements indicate that 30–50% of the total nitrogen (N) loss in the ocean occurs in oxygen minimum zones (OMZs). This pelagic N-removal takes place within only ∼0.1% of the ocean volume, hence moderate variations in the extent of OMZs due to global warming may have a large impact on the global N-cycle. We examined the effect of oxygen (O2) on anammox, NH3 oxidation and NO3− reduction in 15N-labeling experiments with varying O2 concentrations (0–25 µmol L−1) in the Namibian and Peruvian OMZs. Our results show that O2 is a major controlling factor for anammox activity in OMZ waters. Based on our O2 assays we estimate the upper limit for anammox to be ∼20 µmol L−1. In contrast, NH3 oxidation to NO2− and NO3− reduction to NO2− as the main NH4+ and NO2− sources for anammox were only moderately affected by changing O2 concentrations. Intriguingly, aerobic NH3 oxidation was active at non-detectable concentrations of O2, while anaerobic NO3− reduction was fully active up to at least 25 µmol L−1 O2. Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O2 concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O2-sensitivity of anammox itself, and not by any effects of O2 on the tightly coupled pathways of aerobic NH3 oxidation and NO3− reduction. With anammox bacteria in the marine environment being active at O2 levels ∼20 times higher than those known to inhibit their cultured counterparts, the oceanic volume potentially acting as a N-sink increases tenfold. The predicted expansion of OMZs may enlarge this volume even further. Our study provides the first robust estimates of O2 sensitivities for processes directly and indirectly connected with N-loss. These are essential to assess the effects of ocean de-oxygenation on oceanic N-cycling

Isolation and Physiological Characterization of Psychrophilic Denitrifying Bacteria from Permanently Cold Arctic Fjord Sediments (Svalbard, Norway)

A large proportion of reactive nitrogen loss from polar sediments is mediated by denitrification, but microorganisms mediating denitrification in polar environments remain poorly characterized. A combined approach of most-probable-number (MPN) enumeration, cultivation and physiological characterization was used to describe psychrophilic denitrifying bacterial communities in sediments of three Arctic fjords in Svalbard (Norway). A MPN assay showed the presence of 10(sup 3)10(sup 6) cells of psychrophilic nitrate-respiring bacteria g(sup 1) of sediment. Fifteen strains within the Proteobacteria were isolated using a systematic enrichment approach with organic acids as electron donors and nitrate as an electron acceptor. Isolates belonged to five genera, including Shewanella, Pseudomonas, Psychromonas (Gammaproteobacteria), Arcobacter (Epsilonproteobacteria) and Herminiimonas (Betaproteobacteria). All isolates were denitrifiers, except Shewanella, which exhibited the capacity for dissimilatory nitrate reduction to ammonium (DNRA). Growth from 0 to 40 degC demonstrated that all genera except Shewanella were psychrophiles with optimal growth below 15 degC, and adaptation to low temperature was demonstrated as a shift from primarily C16:0 saturated fatty acids to C16:1 monounsaturated fatty acids at lower temperatures. This study provides the first targeted enrichment and characterization of psychrophilic denitrifying bacteria from polar sediments, and two genera, Arcobacter and Herminiimonas, are isolated for the first time from permanently cold marine sediments

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

Regulation of benthic oxygen fluxes in permeable sediments of the coastal ocean

Large areas of the oceanic shelf are composed of sandy sediments through which reactive solutes are transported via porewater advection fueling active microbial communities. The advective oxygen transport in permeable sands of the North Sea was investigated under in situ conditions using a new benthic observatory to assess the dynamic interaction of hydrodynamics, sediment morphodynamics, and oxygen penetration depth. During 16 deployments, concurrent measurement of current velocity, sediment topography, and porewater oxygen concentration were carried out. In all cases the oxyclines were found at depths of 1–6 cm, correlating with the topography of stationary and migrating bedforms (ripples). Different conditions in terms of bottom water currents and bedform migration led to fluctuating oxygen penetration depths and, hence, highly variable redox conditions in up to 2.5 cm thick layers beneath the surface. Volumetric oxygen consumption rates of surface sediments were measured on board in flow-through reactors. Bedform migration was found to reduce consumption rates by up to 50%, presumably caused by the washout of organic carbon that is otherwise trapped in the pore space of the sediment. Based on the observations we found oxygen penetration depths to be largely controlled by oxygen consumption rates, grain size, and current velocity. These controlling variables are summarized by an adapted Damköhler number which allows for prediction of oxygen penetretion depths based on a simple scaling law. By integrating the oxygen consumption rates over the oxygen penetration depth, oxygen fluxes of 8–34 mmol m−2 d−1 were estimate

The impact of bedform migration on benthic oxygen fluxes

Permeable sediments are found wide spread in river beds and on continental shelves. The transport of these sediments is forced by bottom water currents and leads to the formation of bedforms such as ripples and dunes. The bottom water flow across the bedforms results in pressure gradients that drive pore water flow within the permeable sediment and enhance the supply of reactive substrates for biogeochemical processes. This transport-reaction system has been extensively studied for the case of stationary bedforms, whereas bedform migration—the most ubiquitous form of sediment transport—has been often ignored. To study the impact of sediment transport on pore water flow, we incorporated an empirical model of bedform migration into a numerical transport-reaction model for porous media, using oxygen as reactive solute. The modeled oxygen flux changes significantly as soon as the sediment divides into an upper mobile layer (migrating bedform) and a stationary layer underneath. The bedform is increasingly flushed with oxic bottom water, whereas pressure gradients and pore water flow reverse at increasing rate underneath the bedform. This suppresses net pore water displacement and reduces the oxygen penetration depth up to 90%. In effect, the overall oxygen uptake decreases significantly with bedform migration although bottom water velocities increase. This counterintuitive effect is systematically described for a range of different sediment types, current velocities, and respiration rates and should be considered in future studies

Enhanced Nitrogen Loss by Eddy-Induced Vertical Transport in the Offshore Peruvian Oxygen Minimum Zone

The eastern tropical South Pacific (ETSP) upwelling region is one of the ocean’s largest sinks of fixed nitrogen, which is lost as N2 via the anaerobic processes of anammox and denitrification. One-third of nitrogen loss occurs in productive shelf waters stimulated by organic matter export as a result of eastern boundary upwelling. Offshore, nitrogen loss rates are lower, but due to its sheer size this area accounts for ~70% of ETSP nitrogen loss. How nitrogen loss and primary production are regulated in the offshore ETSP region where coastal upwelling is less influential remains unclear. Mesoscale eddies, ubiquitous in the ETSP region, have been suggested to enhance vertical nutrient transport and thereby regulate primary productivity and hence organic matter export. Here, we investigated the impact of mesoscale eddies on anammox and denitrification activity using 15N-labelled in situ incubation experiments. Anammox was shown to be the dominant nitrogen loss process, but varied across the eddy, whereas denitrification was below detection at all stations. Anammox rates at the eddy periphery were greater than at the center. Similarly, depth-integrated chlorophyll paralleled anammox activity, increasing at the periphery relative to the eddy center; suggestive of enhanced organic matter export along the periphery supporting nitrogen loss. This can be attributed to enhanced vertical nutrient transport caused by an eddy-driven submesoscale mechanism operating at the eddy periphery. In the ETSP region, the widespread distribution of eddies and the large heterogeneity observed in anammox rates from a compilation of stations suggests that eddy-driven vertical nutrient transport may regulate offshore primary production and thereby nitrogen loss