Hannah K. Marchant

The fate of nitrate in intertidal permeable sediments

Coastal zones act as a sink for riverine and atmospheric nitrogen inputs and thereby buffer the open ocean from the effects of anthropogenic activity. Recently, microbial activity in sandy permeable sediments has been identified as a dominant source of N-loss in coastal zones, namely through denitrification. Some of the highest coastal denitrification rates measured so far occur within the intertidal permeable sediments of the eutrophied Wadden Sea. Still, denitrification alone can often account for only half of the substantial nitrate (NO3 −) consumption. Therefore, to investigate alternative NO3 − sinks such as dissimilatory nitrate reduction to ammonium (DNRA), intracellular nitrate storage by eukaryotes and isotope equilibration effects we carried out 15NO3 − amendment experiments. By considering all of these sinks in combination, we could quantify the fate of the 15NO3 − added to the sediment. Denitrification was the dominant nitrate sink (50–75%), while DNRA, which recycles N to the environment accounted for 10–20% of NO3 − consumption. Intriguingly, we also observed that between 20 and 40% of 15NO3 − added to the incubations entered an intracellular pool of NO3 − and was subsequently respired when nitrate became limiting. Eukaryotes were responsible for a large proportion of intracellular nitrate storage, and it could be shown through inhibition experiments that at least a third of the stored nitrate was subsequently also respired by eukaryotes. The environmental significance of the intracellular nitrate pool was confirmed by in situ measurements which revealed that intracellular storage can accumulate nitrate at concentrations six fold higher than the surrounding porewater. This intracellular pool is so far not considered when modeling N-loss from intertidal permeable sediments; however it can act as a reservoir for nitrate during low tide. Consequently, nitrate respiration supported by intracellular nitrate storage can add an additional 20% to previous nitrate reduction estimates in intertidal sediments, further increasing their contribution to N-loss.

The microbial nitrogen-cycling network

Nitrogen is an essential component of all living organisms and the main nutrient limiting life on our planet. By far, the largest inventory of freely accessible nitrogen is atmospheric dinitrogen, but most organisms rely on more bioavailable forms of nitrogen, such as ammonium and nitrate, for growth. The availability of these substrates depends on diverse nitrogen-transforming reactions that are carried out by complex networks of metabolically versatile microorganisms. In this Review, we summarize our current understanding of the microbial nitrogen-cycling network, including novel processes, their underlying biochemical pathways, the involved microorganisms, their environmental importance and industrial applications.

Defences against oxidative stress in vibrios associated with corals.

Bacteria colonizing healthy coral tissue may produce enzymes capable of overcoming the toxic effects of reactive oxygen species, including superoxide dismutase (SOD) and catalase. Significant differences in the activities of these enzymes were observed in cultures of Vibrio campbellii, Vibrio coralliilyticus, Vibrio harveyi, Vibrio mediterranei, Vibrio pelagius, Vibrio rotiferanus, Vibrio tasmaniensis, and Photobacterium eurosenbergii isolated from healthy, bleached or necrotic tropical and cold water corals. Levels of SOD in exponential phase cultures of V. coralliilyticus grown at 28 degrees C were only slightly higher than those grown at 16 degrees C whereas the levels in stationary phase cultures at 28 degrees C were 7.3 x higher than those at 16 degrees C. The increase in catalase activity of V. coralliilyticus and V. harveyi upon entry to stationary phase conferred protection against killing by oxidative stress. Increased temperature affected up-regulation of enzymes in stationary phase cultures, but pretreatment of cultures with hydrogen peroxide had no significant effect on induction of catalase or SOD. The increased activities appear to be due to up-regulation of gene expression rather than induction of different forms of the enzymes. We suggest that SOD and catalase are unlikely to be major factors in the virulence of these bacteria for corals and that their main function may be to protect against endogenous superoxide.

Simple approach for the preparation of 15− 15N2-enriched water for nitrogen fixation assessments: evaluation, application and recommendations

Recent findings revealed that the commonly used 15N2 tracer assay for the determination of dinitrogen (N2) fixation can underestimate the activity of aquatic N2-fixing organisms. Therefore, a modification to the method using pre-prepared 15−15N2-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of 15−15N2-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add 15−15N2 gas to the water in amounts exceeding the standard N2 solubility, followed by vigorous agitation (vortex mixing ≥ 5 min). Optionally, water can be degassed at low-pressure (≥950 mbar) for 10 min prior to the 15−15N2 gas addition to indirectly enhance the 15−15N2 concentration. This preparation of 15−15N2-enriched water can be done within 1 h using standard laboratory equipment. The final 15N-atom% excess was 5% after replacing 2–5% of the incubation volume with 15−15N2-enriched water. Notably, the addition of 15−15N2-enriched water can alter levels of trace elements in the incubation water due to the contact of 15−15N2-enriched water with glass, plastic and rubber ware. In our tests, levels of trace elements (Fe, P, Mn, Mo, Cu, Zn) increased by up to 0.1 nmol L−1 in the final incubation volume, which may bias rate measurements in regions where N2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with 15−15N2. The 15−15N2 was injected as a bubble directly to the incubation water, followed by gentle shaking. Immediately thereafter, the bubble was replaced with water to stop the 15−15N2 equilibration. This approach achieved a 15N-atom% excess of 6.6 ± 1.7% when adding 2 mL 15−15N2 per liter of incubation water. The herein presented methodological tests offer guidelines for the 15N2 tracer assay and thus, are crucial to circumvent methodological draw-backs for future N2 fixation assessments.

Denitrifying community in coastal sediments performs aerobic and anaerobic respiration simultaneously

Nitrogen (N) input to the coastal oceans has increased considerably because of anthropogenic activities, however, concurrent increases have not occurred in open oceans. It has been suggested that benthic denitrification in sandy coastal sediments is a sink for this N. Sandy sediments are dynamic permeable environments, where electron acceptor and donor concentrations fluctuate over short temporal and spatial scales. The response of denitrifiers to these fluctuations are largely unknown, although previous observations suggest they may denitrify under aerobic conditions. We examined the response of benthic denitrification to fluctuating oxygen concentrations, finding that denitrification not only occurred at high O2 concentrations but was stimulated by frequent switches between oxic and anoxic conditions. Throughout a tidal cycle, in situtranscription of genes for aerobic respiration and denitrification were positively correlated within diverse bacterial classes, regardless of O2 concentrations, indicating that denitrification gene transcription is not strongly regulated by O2 in sandy sediments. This allows microbes to respond rapidly to changing environmental conditions, but also means that denitrification is utilized as an auxiliary respiration under aerobic conditions when imbalances occur in electron donor and acceptor supply. Aerobic denitrification therefore contributes significantly to N-loss in permeable sediments making the process an important sink for anthropogenic N-inputs.

Recent advances in marine N-cycle studies using 15N labeling methods

15N enriched compounds such as ammonium and nitrate, as well as 15-15N2 gas are invaluable tools in marine N-cycle research. 15N stable isotope approaches allow researchers to delve into the often complex world of N-transformations and trace microbially mediated processes such as nitrification, denitrification, anammox and N-fixation. While 15N stable isotope approaches are well established, experimental approaches which take advantage of them are constantly evolving. Here we summarize recent advances in methodology, including in the direct application of 15N stable isotopes themselves, improved experimental design and the use of 15N stable isotopes in single cell studies. Furthermore, we discuss how these advances have led to new insights into marine N-cycling, particularly in the fields of nitrification and N-fixation.

High rates of microbial dinitrogen fixation and sulfate reduction associated with the Mediterranean seagrass Posidonia oceanica

Seagrass meadows of Posidonia oceanica represent hotspots of productivity in the oligotrophic Mediterranean Sea. The lack of dissolved inorganic nitrogen (DIN) in the seawater suggests that the N-demand of these meadows might be in part supported by microbial dinitrogen (N2) fixation. However, currently there are no direct N2 fixation measurements available for this productive marine macrophyte. Here we investigated N2 fixation activity associated with P. oceanica leaf, rhizome and root pieces. In 15N2 incubations, the roots exhibited highest rates of N2 fixation. The rates varied considerably between replicates, presumably due to a patchy microbial colonization of the roots. Additions of organic carbon compounds (acetate, glucose, sucrose or algal lysate) did not enhance the N2 fixation rates. Sulfate reduction rates measured alongside were also highest in root incubations. Correspondingly, sequences of the nifH gene (a marker gene for the iron protein of the N2-fixing enzyme nitrogenase) related to known sulfate-reducing bacteria were retrieved from P. oceanica roots. Other nifH sequences clustered with known heterotrophic diazotrophs previously identified in other marine macrophytes. In particular, many sequences obtained from P. oceanica roots were similar (>94%) to a saltmarsh rhizosphere-associated heterotrophic diazotroph, indicating that heterotrophic lifestyle might be common among marine macrophyte-associated diazotrophs.

High single cell diversity in carbon and nitrogen assimilation by a chain-forming diatom across a century: Single cell diversity in Skeletonema

Summary Almost a century ago Redfield discovered a relatively constant ratio between carbon, nitrogen and phosphorus in particulate organic matter and nitrogen and phosphorus of dissolved nutrients in seawater. Since then, the riverine export of nitrogen to the ocean has increased 20 fold. High abundance of resting stages in sediment layers dated more than a century back indicate that the common planktonic diatom Skeletonema marinoi has endured this eutrophication. We germinated unique genotypes from resting stages originating from isotope‐dated sediment layers (15 and 80 years old) in a eutrophied fjord. Using secondary ion mass spectrometry (SIMS) combined with stable isotopic tracers, we show that the cell‐specific carbon and nitrogen assimilation rates vary by an order of magnitude on a single‐cell level but are significantly correlated during the exponential growth phase, resulting in constant assimilation quota in cells with identical genotypes. The assimilation quota varies largely between different clones independent of age. We hypothesize that the success of S. marinoi in coastal waters may be explained by its high diversity of nutrient demand not only at a clone‐specific level but also at the single‐cell level, whereby the population can sustain and adapt to dynamic nutrient conditions in the environment.

Metabolic specialization of denitrifiers in permeable sediments controls N2O emissions: Metabolic specialization & N2O emissions in sands

Coastal oceans receive large amounts of anthropogenic fixed nitrogen (N), most of which is denitrified in the sediment before reaching the open ocean. Sandy sediments, which are common in coastal regions, seem to play an important role in catalysing this N-loss. Permeable sediments are characterized by advective porewater transport, which supplies high fluxes of organic matter into the sediment, but also leads to fluctuations in oxygen and nitrate concentrations. Little is known about how the denitrifying communities in these sediments are adapted to such fluctuations. Our combined results indicate that denitrification in eutrophied sandy sediments from the worlds largest tidal flat system, the Wadden Sea, is carried out by different groups of microorganisms. This segregation leads to the formation of N2 O which is advectively transported to the overlying waters and thereby emitted to the atmosphere. At the same time, the production of N2 O within the sediment supports a subset of Flavobacteriia which appear to be specialized on N2 O reduction. If the mechanisms shown here are active in other coastal zones, then denitrification in eutrophied sandy sediments may substantially contribute to current marine N2 O emissions.