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We describe diazotrophs present during a 2015 GEOTRACES expedition through the Canadian Arctic Gateway (CAG) using nifH metabarcoding. In the less studied Labrador Sea, Bradyrhizobium sp. and Vitreoscilla sp. nifH variants were dominant, while in Baffin Bay, a Stutzerimonas stutzeri variant was dominant. In comparison, the Canadian Arctic Archipelago (CAA) was characterized by a broader set of dominant variants belonging to Desulfobulbaceae, Desulfuromonadales, Arcobacter sp., Vibrio spp., and Sulfuriferula sp. Although dominant diazotrophs fell within known nifH clusters I and III, only a few of these variants were frequently recovered in a 5-year weekly nifH times series in the coastal NW Atlantic presented herein, notably S. stutzeri and variants belonging to Desulfobacterales and Desulfuromonadales. In addition, the majority of dominant Arctic nifH variants shared low similarity (< 92% nucleotide identities) to sequences in a global noncyanobacterial diazotroph catalog recently compiled by others. We further detected UCYN-A throughout the CAG at low-levels using quantitative-PCR assays. Temperature, depth, salinity, oxygen, and nitrate were most strongly correlated to the Arctic diazotroph diversity observed, and we found a stark division between diazotroph communities of the Labrador Sea versus Baffin Bay and the CAA, hence establishing that a previously unknown biogeographic community division can occur for diazotrophs in the CAG., (© The Author(s) 2023. Published by Oxford University Press on behalf of FEMS.)

We present the first geomicrobiological characterization of the meromictic water column of Powell Lake (British Columbia, Canada), a former fjord, which has been stably stratified since the last glacial period. Its deepest layers (300-350 m) retain isolated, relict seawater from that period. Fine-scale vertical profiling of the water chemistry and microbial communities allowed subdivision of the water column into distinct geomicrobiological zones. These zones were further characterized by phylogenetic and functional marker genes from amplicon and shotgun metagenome sequencing. Binning of metagenomic reads allowed the linkage of function to specific taxonomic groups. Statistical analyses (analysis of similarities, Bray-Curtis similarity) confirmed that the microbial community structure followed closely the geochemical zonation. Yet, our characterization of the genetic potential relevant to carbon, nitrogen and sulphur cycling of each zone revealed unexpected features, including potential for facultative anaerobic methylotrophy, nitrogen fixation despite high ammonium concentrations and potential micro-aerobic nitrifiers within the chemocline. At the oxic-suboxic interface, facultative anaerobic potential was found in the widespread freshwater lineage acI (Actinobacteria), suggesting intriguing ecophysiological similarities to the marine SAR11. Evolutionary divergent lineages among diverse phyla were identified in the ancient seawater zone and may indicate novel adaptations to this unusual environment., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Symbiotic relationships between phytoplankton and N2-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N2) fixation and primary production by the UCYN-A-haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N2 fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO2 fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO2 fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N2 fixation and the transfer of N2-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.

Unicellular, diazotrophic cyanobacteria temporally separate dinitrogen (N2) fixation and photosynthesis to prevent inactivation of the nitrogenase by oxygen. This temporal segregation is regulated by a circadian clock with oscillating activities of N2 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 N2 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 N2 fixation and photosynthesis commonly observed. However, N2 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.

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 from ocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogen budget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 Tg N yr−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 method can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimates N2-fixation rates by an average of 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 γ-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 ± 1 Tg N yr−1 and 24 ±1 Tg N yr−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 ± 8 Tg N yr−1 to 177 ± 8 Tg N yr−1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought.

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 (O(2)) on anammox, NH(3) oxidation and NO(3)(-) reduction in (15)N-labeling experiments with varying O(2) concentrations (0-25 µmol L(-1)) in the Namibian and Peruvian OMZs. Our results show that O(2) is a major controlling factor for anammox activity in OMZ waters. Based on our O(2) assays we estimate the upper limit for anammox to be ~20 µmol L(-1). In contrast, NH(3) oxidation to NO(2)(-) and NO(3)(-) reduction to NO(2)(-) as the main NH(4)(+) and NO(2)(-) sources for anammox were only moderately affected by changing O(2) concentrations. Intriguingly, aerobic NH(3) oxidation was active at non-detectable concentrations of O(2), while anaerobic NO(3)(-) reduction was fully active up to at least 25 µmol L(-1) O(2). Hence, aerobic and anaerobic N-cycle pathways in OMZs can co-occur over a larger range of O(2) concentrations than previously assumed. The zone where N-loss can occur is primarily controlled by the O(2)-sensitivity of anammox itself, and not by any effects of O(2) on the tightly coupled pathways of aerobic NH(3) oxidation and NO(3)(-) reduction. With anammox bacteria in the marine environment being active at O(2) 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 O(2) 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., (© 2011 Kalvelage et al.)

Growth, protein amount, and activity levels of metabolic pathways in Trichodesmium are influenced by environmental changes such as elevated pCO(2) and temperature. This study examines changes in the expression of essential metabolic genes in Trichodesmium grown under a matrix of pCO(2) (400 and 900 µatm) and temperature (25 and 31°C). Using RT-qPCR, we studied 21 genes related to four metabolic functional groups: CO(2) concentrating mechanism (bicA1, bicA2, ccmM, ccmK2, ccmK3, ndhF4, ndhD4, ndhL, chpX), energy metabolism (atpB, sod, prx, glcD), nitrogen metabolism (glnA, hetR, nifH), and inorganic carbon fixation and photosynthesis (rbcL, rca, psaB, psaC, psbA). nifH and most photosynthetic genes exhibited relatively high abundance and their expression was influenced by both environmental parameters. A two to three orders of magnitude increase was observed for glnA and hetR only when both pCO(2) and temperature were elevated. CO(2) concentrating mechanism genes were not affected by pCO(2) and temperature and their expression levels were markedly lower than that of the nitrogen metabolism and photosynthetic genes. Many of the CO(2) concentrating mechanism genes were co-expressed throughout the day. Our results demonstrate that in Trichodesmium, CO(2) concentrating mechanism genes are constitutively expressed. Co-expression of genes from different functional groups were frequently observed during the first half of the photoperiod when oxygenic photosynthesis and N(2) fixation take place, pointing at the tight and complex regulation of gene expression in Trichodesmium. Here we provide new data linking environmental changes of pCO(2) and temperature to gene expression in Trichodesmium. Although gene expression indicates an active metabolic pathway, there is often an uncoupling between transcription and enzyme activity, such that transcript level cannot usually be directly extrapolated to metabolic activity.

The two commonly applied methods to assess dinitrogen (N(2)) fixation rates are the (15)N(2)-tracer addition and the acetylene reduction assay (ARA). Discrepancies between the two methods as well as inconsistencies between N(2) fixation rates and biomass/growth rates in culture experiments have been attributed to variable excretion of recently fixed N(2). Here we demonstrate that the (15)N(2)-tracer addition method underestimates N(2) fixation rates significantly when the (15)N(2) tracer is introduced as a gas bubble. The injected (15)N(2) gas bubble does not attain equilibrium with the surrounding water leading to a (15)N(2) concentration lower than assumed by the method used to calculate (15)N(2)-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 N(2) fixation patterns. Here, we propose and test a modified (15)N(2) tracer method based on the addition of (15)N(2)-enriched seawater that provides an instantaneous, constant enrichment and allows more accurate calculation of N(2) fixation rates for both field and laboratory studies. We hypothesise that application of N(2) fixation measurements using this modified method will significantly reduce the apparent imbalances in the oceanic fixed-nitrogen budget.

We examined the influence of forecasted changes in global temperatures and pCO(2) on N(2) fixation and assimilation in the ecologically important cyanobacterium Trichodesmium spp. Changes of mRNA transcripts (nifH, glnA, hetR, psbA, psaB), protein (nitrogenase, glutamine synthetase) pools and enzymatic activity (nitrogenase) were measured under varying pCO(2) and temperatures. High pCO(2) shifted transcript patterns of all genes, resulting in a more synchronized diel expression. Under the same conditions, we did not observe any significant changes in the protein pools or in total cellular allocations of carbon and nitrogen (i.e. C : N ratio remained stable). Independently of temperature, high pCO(2) (900 microatm) elevated N(2) fixation rates. Levels of the key enzymes, nitrogenase and glutamine synthetase that mediate nitrogen assimilation did not increase, implying that the high pCO(2) allowed higher reaction turnover rates through these key enzymes. Moreover, increased temperatures and high pCO(2) resulted in higher C : P ratios. The plasticity in phosphorous stoichiometry combined with higher enzymatic efficiencies lead to higher growth rates. In cyanobacteria photosynthesis, carbon uptake, respiration, N(2) fixation and nitrogen assimilation share cellular components. We propose that shifted cellular resource and energy allocation among those components will enable Trichodesmium grown at elevated temperatures and pCO(2) to extend its niche in the future ocean, through both tolerance of a broader temperature range and higher P plasticity.

We examined the diel variation in nitrogen and carbon metabolism in Crocosphaera watsonii WH8501 at the physiological and gene expression level in order to determine the temporal constraints for N2 fixation and photosynthesis. N2 fixation and photosynthesis were restricted to the dark and light periods, respectively, during a 24 h light-dark cycle. All genes studied here except one (psbA2) showed diel variations in their expression levels. The highest variation was seen in nifH and nifX relative transcript abundance with a factor of 3-5x10(3) between light and dark periods. Photosynthesis genes showed less variation with a maximum factor of about 500 and always had high relative transcript abundances relative to other genes. At the protein level, the photosystems appeared more stable than the nitrogenase complex over a 24 h light-dark cycle, suggesting that C. watsonii retains the ability to photosynthesize during the dark period of the diel cycle. In contrast, nitrogenase is synthesized daily and exhibits peak abundance during the dark period. Our results have implications for field studies with respect to the interpretation of environmental gene expression data.

The nitrogen cycling of Lake Cadagno was investigated by using a combination of biogeochemical and molecular ecological techniques. In the upper oxic freshwater zone inorganic nitrogen concentrations were low (up to approximately 3.4 microM nitrate at the base of the oxic zone), while in the lower anoxic zone there were high concentrations of ammonium (up to 40 microM). Between these zones, a narrow zone was characterized by no measurable inorganic nitrogen, but high microbial biomass (up to 4 x 10(7) cells ml(-1)). Incubation experiments with (15)N-nitrite revealed nitrogen loss occurring in the chemocline through denitrification (approximately 3 nM N h(-1)). At the same depth, incubations experiments with (15)N(2)- and (13)C(DIC)-labelled bicarbonate, indicated substantial N(2) fixation (31.7-42.1 pM h(-1)) and inorganic carbon assimilation (40-85 nM h(-1)). Catalysed reporter deposition fluorescence in situ hybridization (CARD-FISH) and sequencing of 16S rRNA genes showed that the microbial community at the chemocline was dominated by the phototrophic green sulfur bacterium Chlorobium clathratiforme. Phylogenetic analyses of the nifH genes expressed as mRNA revealed a high diversity of N(2) fixers, with the highest expression levels right at the chemocline. The majority of N(2) fixers were related to Chlorobium tepidum/C. phaeobacteroides. By using Halogen In Situ Hybridization-Secondary Ion Mass Spectroscopy (HISH-SIMS), we could for the first time directly link Chlorobium to N(2) fixation in the environment. Moreover, our results show that N(2) fixation could partly compensate for the N loss and that both processes occur at the same locale at the same time as suggested for the ancient Ocean.

Increasing quantities of atmospheric anthropogenic fixed nitrogen entering the open ocean could account for up to about a third of the ocean's external (nonrecycled) nitrogen supply and up to approximately 3% of the annual new marine biological production, approximately 0.3 petagram of carbon per year. This input could account for the production of up to approximately 1.6 teragrams of nitrous oxide (N2O) per year. Although approximately 10% of the ocean's drawdown of atmospheric anthropogenic carbon dioxide may result from this atmospheric nitrogen fertilization, leading to a decrease in radiative forcing, up to about two-thirds of this amount may be offset by the increase in N2O emissions. The effects of increasing atmospheric nitrogen deposition are expected to continue to grow in the future.

The marine autotroph Aureococcus anophagefferens (Pelagophyceae) was rendered axenic in order to investigate hydrolysis rates of peptides, chitobiose, acetamide, and urea as indicators of the ability to support growth on dissolved organic nitrogen. Specific rates of hydrolysis varied between 8 and 700% of rates observed in associated heterotrophic marine bacteria.

We report a new synthesis of best estimates of the inputs of fixed nitrogen to the world ocean via atmospheric deposition and compare this to fluvial inputs and dinitrogen fixation. We evaluate the scale of human perturbation of these fluxes. Fluvial inputs dominate inputs to the continental shelf, and we estimate that about 75% of this fluvial nitrogen escapes from the shelf to the open ocean. Biological dinitrogen fixation is the main external source of nitrogen to the open ocean, i.e., beyond the continental shelf. Atmospheric deposition is the primary mechanism by which land-based nitrogen inputs, and hence human perturbations of the nitrogen cycle, reach the open ocean. We estimate that anthropogenic inputs are currently leading to an increase in overall ocean carbon sequestration of ~0.4% (equivalent to an uptake of 0.15 Pg C yr−1 and less than the Duce et al. (2008) estimate). The resulting reduction in climate change forcing from this ocean CO2 uptake is offset to a small extent by an increase in ocean N2O emissions. We identify four important feedbacks in the ocean atmosphere nitrogen system that need to be better quantified to improve our understanding of the perturbation of ocean biogeochemistry by atmospheric nitrogen inputs. These feedbacks are recycling of (1) ammonia and (2) organic nitrogen from the ocean to the atmosphere and back, (3) the suppression of nitrogen fixation by increased nitrogen concentrations in surface waters from atmospheric deposition, and (4) increased loss of nitrogen from the ocean by denitrification due to increased productivity stimulated by atmospheric inputs. [ABSTRACT FROM AUTHOR]

Nitrogen-doped ZnO films were grown by metal organic chemical vapor deposition using diallylamine as the doping source. Secondary ion mass spectrometry measurements attest for the incorporation of nitrogen up to 1020 cm-3, and both photoluminescence and Raman characterization exhibit nitrogen related peaks. As-deposited layers have been characterized by Hall effect, and most exhibit n-type conductivity, with electron concentrations from 1017 up to a few 1019 cm-3. Unstable p-type behavior is sometimes observed on low quality layers grown using a very low RVI/II ratio. Ex situ annealing experiments in oxygen atmosphere show different tendencies depending on the growth conditions and can lead to a strong decrease in the electron concentration by three orders of magnitude. These results are discussed in relation to the structural and optical characterizations. [ABSTRACT FROM AUTHOR]

Diazotrophic (N2-fixing) cyanobacteria provide the biological source of new nitrogen for large parts of the ocean. However, little is known about their sensitivity to global change. Here we show that the single most important nitrogen fixer in today's ocean, Trichodesmium, is strongly affected by changes in CO2 concentrations. Cell division rate doubled with rising CO2 (glacial to projected year 2100 levels) prompting lower carbon, nitrogen and phosphorus cellular contents, and reduced cell dimensions. N2 fixation rates per unit of phosphorus utilization as well as C:P and N:P ratios more than doubled at high CO2, with no change in C:N ratios. This could enhance the productivity of N-limited oligotrophic oceans, drive some of these areas into P limitation, and increase biological carbon sequestration in the ocean. The observed CO2 sensitivity of Trichodesmium could thereby provide a strong negative feedback to atmospheric CO2 increase. [ABSTRACT FROM AUTHOR]