Isabell Klawonn

Meiofauna increases bacterial denitrification in marine sediments

Denitrification is a critical process that can alleviate the effects of excessive nitrogen availability in aquatic ecosystems subject to eutrophication. An important part of denitrification occurs in benthic systems where bioturbation by meiofauna (invertebrates <1 mm) and its effect on element cycling are still not well understood. Here we study the quantitative impact of meiofauna populations of different abundance and diversity, in the presence and absence of macrofauna, on nitrate reduction, carbon mineralization and methane fluxes. In sediments with abundant and diverse meiofauna, denitrification is double that in sediments with low meiofauna, suggesting that meiofauna bioturbation has a stimulating effect on nitrifying and denitrifying bacteria. However, high meiofauna densities in the presence of bivalves do not stimulate denitrification, while dissimilatory nitrate reduction to ammonium rate and methane efflux are significantly enhanced. We demonstrate that the ecological interactions between meio-, macrofauna and bacteria are important in regulating nitrogen cycling in soft-sediment ecosystems.

Aerobic and anaerobic nitrogen transformation processes in N-2-fixing cyanobacterial aggregates

Colonies of N2-fixing cyanobacteria are key players in supplying new nitrogen to the ocean, but the biological fate of this fixed nitrogen remains poorly constrained. Here, we report on aerobic and anaerobic microbial nitrogen transformation processes that co-occur within millimetre-sized cyanobacterial aggregates (Nodularia spumigena) collected in aerated surface waters in the Baltic Sea. Microelectrode profiles showed steep oxygen gradients inside the aggregates and the potential for nitrous oxide production in the aggregates’ anoxic centres. 15N-isotope labelling experiments and nutrient analyses revealed that N2 fixation, ammonification, nitrification, nitrate reduction to ammonium, denitrification and possibly anaerobic ammonium oxidation (anammox) can co-occur within these consortia. Thus, N. spumigena aggregates are potential sites of nitrogen gain, recycling and loss. Rates of nitrate reduction to ammonium and N2 were limited by low internal nitrification rates and low concentrations of nitrate in the ambient water. Presumably, patterns of N-transformation processes similar to those observed in this study arise also in other phytoplankton colonies, marine snow and fecal pellets. Anoxic microniches, as a pre-condition for anaerobic nitrogen transformations, may occur within large aggregates (⩾1 mm) even when suspended in fully oxygenated waters, whereas anoxia in small aggregates (<1 to ⩾0.1 mm) may only arise in low-oxygenated waters (⩽25 μM). We propose that the net effect of aggregates on nitrogen loss is negligible in NO3−-depleted, fully oxygenated (surface) waters. In NO3−-enriched (>1.5 μM), O2-depleted water layers, for example, in the chemocline of the Baltic Sea or the oceanic mesopelagic zone, aggregates may promote N-recycling and -loss processes.

N2-fixation, ammonium release and N-transfer to the microbial and classical food web within a plankton community

We investigated the role of N2-fixation by the colony-forming cyanobacterium, Aphanizomenon spp., for the plankton community and N-budget of the N-limited Baltic Sea during summer by using stable isotope tracers combined with novel secondary ion mass spectrometry, conventional mass spectrometry and nutrient analysis. When incubated with 15N2, Aphanizomenon spp. showed a strong 15N-enrichment implying substantial 15N2-fixation. Intriguingly, Aphanizomenon did not assimilate tracers of 15NH4+ from the surrounding water. These findings are in line with model calculations that confirmed a negligible N-source by diffusion-limited NH4+ fluxes to Aphanizomenon colonies at low bulk concentrations (<250 nm) as compared with N2-fixation within colonies. No N2-fixation was detected in autotrophic microorganisms <5 μm, which relied on NH4+ uptake from the surrounding water. Aphanizomenon released about 50% of its newly fixed N2 as NH4+. However, NH4+ did not accumulate in the water but was transferred to heterotrophic and autotrophic microorganisms as well as to diatoms (Chaetoceros sp.) and copepods with a turnover time of ~5 h. We provide direct quantitative evidence that colony-forming Aphanizomenon releases about half of its recently fixed N2 as NH4+, which is transferred to the prokaryotic and eukaryotic plankton forming the basis of the food web in the plankton community. Transfer of newly fixed nitrogen to diatoms and copepods furthermore implies a fast export to shallow sediments via fast-sinking fecal pellets and aggregates. Hence, N2-fixing colony-forming cyanobacteria can have profound impact on ecosystem productivity and biogeochemical processes at shorter time scales (hours to days) than previously thought.

Nitrogen fixation by cyanobacteria stimulates production in Baltic food webs.

Filamentous, nitrogen-fixing cyanobacteria form extensive summer blooms in the Baltic Sea. Their ability to fix dissolved N2 allows cyanobacteria to circumvent the general summer nitrogen limitation, while also generating a supply of novel bioavailable nitrogen for the food web. However, the fate of the nitrogen fixed by cyanobacteria remains unresolved, as does its importance for secondary production in the Baltic Sea. Here, we synthesize recent experimental and field studies providing strong empirical evidence that cyanobacterial nitrogen is efficiently assimilated and transferred in Baltic food webs via two major pathways: directly by grazing on fresh or decaying cyanobacteria and indirectly through the uptake by other phytoplankton and microbes of bioavailable nitrogen exuded from cyanobacterial cells. This information is an essential step toward guiding nutrient management to minimize noxious blooms without overly reducing secondary production, and ultimately most probably fish production in the Baltic Sea.

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.

Chemical microenvironments and single-cell carbon and nitrogen uptake in field-collected colonies of Trichodesmium under different pCO2

Gradients of oxygen (O2) and pH, as well as small-scale fluxes of carbon (C), nitrogen (N) and O2 were investigated under different partial pressures of carbon dioxide (pCO2) in field-collected colonies of the marine dinitrogen (N2)-fixing cyanobacterium Trichodesmium. Microsensor measurements indicated that cells within colonies experienced large fluctuations in O2, pH and CO2 concentrations over a day–night cycle. O2 concentrations varied with light intensity and time of day, yet colonies exposed to light were supersaturated with O2 (up to ~200%) throughout the light period and anoxia was not detected. Alternating between light and dark conditions caused a variation in pH levels by on average 0.5 units (equivalent to 15 nmol l−1 proton concentration). Single-cell analyses of C and N assimilation using secondary ion mass spectrometry (SIMS; large geometry SIMS and nanoscale SIMS) revealed high variability in metabolic activity of single cells and trichomes of Trichodesmium, and indicated transfer of C and N to colony-associated non-photosynthetic bacteria. Neither O2 fluxes nor C fixation by Trichodesmium were significantly influenced by short-term incubations under different pCO2 levels, whereas N2 fixation increased with increasing pCO2. The large range of metabolic rates observed at the single-cell level may reflect a response by colony-forming microbial populations to highly variable microenvironments.

Cell-specific nitrogen- and carbon-fixation of cyanobacteria in a temperate marine system (Baltic Sea).

We analysed N2 - and carbon (C) fixation in individual cells of Baltic Sea cyanobacteria by combining stable isotope incubations with secondary ion mass spectrometry (SIMS). Specific growth rates based on N2 - and C-fixation were higher for cells of Dolichospermum spp. than for Aphanizomenon sp. and Nodularia spumigena. The cyanobacterial biomass, however, was dominated by Aphanizomenon sp., which contributed most to total N2 -fixation in surface waters of the Northern Baltic Proper. N2 -fixation by Pseudanabaena sp. and colonial picocyanobacteria was not detectable. N2 -fixation by Aphanizomenon sp., Dolichospermum spp. and N. spumigena populations summed up to total N2 -fixation, thus these genera appeared as sole diazotrophs within the Baltic Seas euphotic zone, while their mean contribution to total C-fixation was 21%. Intriguingly, cell-specific N2 -fixation was eightfold higher at a coastal station compared to an offshore station, revealing coastal zones as habitats with substantial N2 -fixation. At the coastal station, the cell-specific C- to N2 -fixation ratio was below the cellular C:N ratio, i.e. N2 was assimilated in excess to C-fixation, whereas the C- to N2 -fixation ratio exceeded the C:N ratio in offshore sampled diazotrophs. Our findings highlight SIMS as a powerful tool not only for qualitative but also for quantitative N2 -fixation assays in aquatic environments.