Dinitrogen fixation in the unicellular diazotroph Crocosphaera watsonii

Crocosphaera watsonii is an abundant organism in the tropical and subtropical ocean and is considered to be an important contributor to the marine nitrogen cycle due to its ability to fix dinitrogen (N2). Light-dark cycles were used to determine the diel variation in N2 fixation and photosynthesis in the unicellular diazotrophic cyanobacterium C. watsonii WH8501. A first set of experiments showed that N2 fixation and photosynthesis were separated temporally with N2 fixation during the dark and photosynthesis during the light periods. Due to the storage of carbon reserves during the day and subsequent respiration at night, C. watsonii links its cellular nitrogen and carbon metabolism. N2 fixation and photosynthesis appear to be regulated and optimized by a circadian rhythm. Gene expression analysis demonstrated cyclic patterns of the major genes involved in nitrogen and carbon metabolism. However, the patterns of expression did not coincide with patterns of activity with the gene expression peaking several hours prior to the activity. In a second set of experiments, N2 fixation and photosynthesis were simultaneously measurable at population level in the respective dark period, i.e. an artificial light period instead of a dark period. Single-cell analysis using nanoSIMS revealed that the co-occurrence of N2 fixation and photosynthesis at population level was probably due to each individual cell’s capability of coping with the O2-sensitivity of the nitrogenase enzyme. In addition, single-cell rates showed large variability with the highest rate about six times higher than the mean rate during the normal dark N2 fixation. The experimental work with a pure culture of a diazotroph provided the opportunity for a re-assessment of the two commonly used methods for the measurement of N2 fixation in both field and laboratory studies, i.e. the 15N2-tracer addition method and the acetylene reduction assay

Methodological Underestimation of Oceanic Nitrogen Fixation Rates

The two commonly applied methods to assess dinitrogen (N2) fixation rates are the 15N2-tracer addition and the acetylene reduction assay (ARA). Discrepancies between the two methods as well as inconsistencies between N2 fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N2. Here we demonstrate that the 15N2-tracer addition method underestimates N2 fixation rates significantly when the 15N2 tracer is introduced as a gas bubble. The injected 15N2 gas bubble does not attain equilibrium with the surrounding water leading to a 15N2 concentration lower than assumed by the method used to calculate 15N2-fixation rates. The resulting magnitude of underestimation varies with the incubation time, to a lesser extent on the amount of injected gas and is sensitive to the timing of the bubble injection relative to diel N2 fixation patterns. Here, we propose and test a modified 15N2 tracer method based on the addition of 15N2-enriched seawater that provides an instantaneous, constant enrichment and allows more accurate calculation of N2 fixation rates for both field and laboratory studies. We hypothesise that application of N2 fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget

No nitrogen fixation in the Bay of Bengal?

The Bay of Bengal (BoB) has long stood as a biogeochemical enigma, with subsurface waters containing extremely low, but persistent, concentrations of oxygen in the nanomolar range which – for some, yet unconstrained, reason – are prevented from becoming anoxic. One reason for this may be the low productivity of the BoB waters due to nutrient limitation and the resulting lack of respiration of organic material at intermediate waters. Thus, the parameters determining primary production are key in understanding what prevents the BoB from developing anoxia. Primary productivity in the sunlit surface layers of tropical oceans is mostly limited by the supply of reactive nitrogen through upwelling, riverine flux, atmospheric deposition, and biological dinitrogen (N2) fixation. In the BoB, a stable stratification limits nutrient supply via upwelling in the open waters, and riverine or atmospheric fluxes have been shown to support only less than one-quarter of the nitrogen for primary production. This leaves a large uncertainty for most of the BoB's nitrogen input, suggesting a potential role of N2 fixation in those waters. Here, we present a survey of N2 fixation and carbon fixation in the BoB during the winter monsoon season. We detected a community of N2 fixers comparable to other oxygen minimum zone (OMZ) regions, with only a few cyanobacterial clades and a broad diversity of non-phototrophic N2 fixers present throughout the water column (samples collected between 10 and 560 m water depth). While similar communities of N2 fixers were shown to actively fix N2 in other OMZs, N2 fixation rates were below the detection limit in our samples covering the water column between the deep chlorophyll maximum and the OMZ. Consistent with this, no N2 fixation signal was visible in δ15N signatures. We suggest that the absence of N2 fixation may be a consequence of a micronutrient limitation or of an O2 sensitivity of the OMZ diazotrophs in the BoB. Exploring how the onset of N2 fixation by cyanobacteria compared to non-phototrophic N2 fixers would impact on OMZ O2 concentrations, a simple model exercise was carried out. We observed that both photic-zone-based and OMZ-based N2 fixation are very sensitive to even minimal changes in water column stratification, with stronger mixing increasing organic matter production and export, which can exhaust remaining O2 traces in the BoB

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

Single-cell imaging of phosphorus uptake shows that key harmful algae rely on different phosphorus sources for growth

Single-cell measurements of biochemical processes have advanced our understanding of cellular physiology in individual microbes and microbial populations. Due to methodological limitations, little is known about single-cell phosphorus (P) uptake and its importance for microbial growth within mixed field populations. Here, we developed a nanometer-scale secondary ion mass spectrometry (nanoSIMS)-based approach to quantify single-cell P uptake in combination with cellular CO2 and N2 fixation. Applying this approach during a harmful algal bloom (HAB), we found that the toxin-producer Nodularia almost exclusively used phosphate for growth at very low phosphate concentrations in the Baltic Sea. In contrast, the non-toxic Aphanizomenon acquired only 15% of its cellular P-demand from phosphate and ~85% from organic P. When phosphate concentrations were raised, Nodularia thrived indicating that this toxin-producer directly benefits from phosphate inputs. The phosphate availability in the Baltic Sea is projected to rise and therefore might foster more frequent and intense Nodularia blooms with a concomitant rise in the overall toxicity of HABs in the Baltic Sea. With a projected increase in HABs worldwide, the capability to use organic P may be a critical factor that not only determines the microbial community structure, but the overall harmfulness and associated costs of algal blooms

A Critical Review of the \u3csup\u3e15\u3c/sup\u3eN\u3csub\u3e2\u3c/sub\u3e Tracer Method to Measure Diazotrophic Production in Pelagic Ecosystems

Dinitrogen (N2) fixation is an important source of biologically reactive nitrogen (N) to the global ocean. The magnitude of this flux, however, remains uncertain, in part because N2 fixation rates have been estimated following divergent protocols and because associated levels of uncertainty are seldom reported—confounding comparison and extrapolation of rate measurements. A growing number of reports of relatively low but potentially significant rates of N2 fixation in regions such as oxygen minimum zones, the mesopelagic water column of the tropical and subtropical oceans, and polar waters further highlights the need for standardized methodological protocols for measurements of N2 fixation rates and for calculations of detection limits and propagated error terms. To this end, we examine current protocols of the 15N2 tracer method used for estimating diazotrophic rates, present results of experiments testing the validity of specific practices, and describe established metrics for reporting detection limits. We put forth a set of recommendations for best practices to estimate N2 fixation rates using 15N2 tracer, with the goal of fostering transparency in reporting sources of uncertainty in estimates, and to render N2 fixation rate estimates intercomparable among studies

Measurement report : Long-range transport and the fate of dimethyl sulfide oxidation products in the free troposphere derived from observations at the high-altitude research station Chacaltaya (5240 m a.s.l.) in the Bolivian Andes

Dimethyl sulfide (DMS) is the primary natural contributor to the atmospheric sulfur burden. Observations concerning the fate of DMS oxidation products after long-range transport in the remote free troposphere are, however, sparse. Here we present quantitative chemical ionization mass spectrometric measurements of DMS and its oxidation products sulfuric acid (H2SO4), methanesulfonic acid (MSA), dimethylsulfoxide (DMSO), dimethylsulfone (DMSO2), methanesulfinic acid (MSIA), methyl thioformate (MTF), methanesulfenic acid (MSEA, CH3SOH), and a compound of the likely structure CH3S(O)(2)OOH in the gas phase, as well as measurements of the sulfate and methanesulfonate aerosol mass fractions. The measurements were performed at the Global Atmosphere Watch (GAW) station Chacaltaya in the Bolivian Andes located at 5240 m above sea level (a.s.l.). DMS and DMS oxidation products are brought to the Andean high-altitude station by Pacific air masses during the dry season after convective lifting over the remote Pacific ocean to 6000-8000 m a.s.l. and subsequent longrange transport in the free troposphere (FT). Most of the DMS reaching the station is already converted to the rather unreactive sulfur reservoirs DMSO2 in the gas phase and methanesulfonate (MS-) in the particle phase, which carried nearly equal amounts of sulfur to the station. The particulate sulfate at Chacaltaya is however dominated by regional volcanic emissions during the time of the measurement and not significantly affected by the marine air masses. In one of the FT events, even some DMS was observed next to reactive intermediates such as methyl thioformate, dimethylsulfoxide, and methanesulfinic acid. Also for this event, back trajectory calculations show that the air masses came from above the ocean (distance > 330 km) with no local surface contacts. This study demonstrates the potential impact of marine DMS emissions on the availability of sulfur containing vapors in the remote free troposphere far away from the ocean.Peer reviewe

Doubling of marine dinitrogen-fixation rates based on direct measurements

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the ocean’s nitrogen inventory and primary productivity. Nitrogen-isotope data fromocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogenbudget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 TgNyr-1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method8 can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimatesN2-fixation rates by an averageof 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and c-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14+/-1TgNyr-1 and 24+/-1TgNyr-1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103+/-8TgNyr-1 to 177+/-8TgNyr-1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought