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Simple approach for the preparation of N-15-15(2)-enriched water for nitrogen fixation assessments: evaluation, application and recommendations
Stockholm University, Faculty of Science, Department of Ecology, Environment and Plant Sciences.
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Number of Authors: 7
2015 (English)In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 6, 769Article in journal (Refereed) Published
Abstract [en]

Recent findings revealed that the commonly used N-15(2) tracer assay for the determination of dinitrogen (N-2) fixation can underestimate the activity of aquatic N-2-fixing organisms. Therefore, a modification to the method using pre-prepared N-15-15(2)-enriched water was proposed. Here, we present a rigorous assessment and outline a simple procedure for the preparation of N-15-15(2)-enriched water. We recommend to fill sterile-filtered water into serum bottles and to add N-15-15(2) gas to the water in amounts exceeding the standard N-2 solubility, followed by vigorous agitation (vortex mixing >= 5 min). Optionally, water can be degassed at low-pressure (>= 950 mbar) for 10 mm prior to the N-15-15(2) gas addition to indirectly enhance the N-15-15(2) concentration. This preparation of N-15-15(2)-enriched water can be done within 1 h using standard laboratory equipment. The final N-15-atom% excess was 5% after replacing 2-5% of the incubation volume with N-15-15(2)-enriched water. Notably, the addition of N-15-15(2)-enriched water can alter levels of trace elements in the incubation water due to the contact of N-15-15(2)-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 N-2 fixation is limited by trace elements. For these regions, we tested an alternative way to enrich water with N-15-15(2). The N-15-15(2) 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 N-15-15(2) equilibration. This approach achieved a N-15-atom% excess of 6.6 +/- 1.7% when adding 2 mL N-15-15(2) per liter of incubation water. The herein presented methodological tests offer guidelines for the N-15(2) tracer assay and thus, are crucial to circumvent methodological draw-backs for future N-2 fixation assessments.

Place, publisher, year, edition, pages
2015. Vol. 6, 769
Keyword [en]
N-2 fixation, cyanobacteria, gas-liquid solution, N-15(2) gas, gas solubility, iron, phosphorus, Nodularia spumigena
National Category
Biological Sciences
Research subject
Marine Ecology
Identifiers
URN: urn:nbn:se:su:diva-120192DOI: 10.3389/fmicb.2015.00769ISI: 000359484100002OAI: oai:DiVA.org:su-120192DiVA: diva2:851331
Available from: 2015-09-04 Created: 2015-09-02 Last updated: 2017-12-04Bibliographically approved
In thesis
1. Marine nitrogen fixation: Cyanobacterial nitrogen fixation and the fate of new nitrogen in the Baltic Sea
Open this publication in new window or tab >>Marine nitrogen fixation: Cyanobacterial nitrogen fixation and the fate of new nitrogen in the Baltic Sea
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Biogeochemical processes in the marine biosphere are important in global element cycling and greatly influence the gas composition of the Earth’s atmosphere. The nitrogen cycle is a key component of marine biogeochemical cycles. Nitrogen is an essential constituent of living organisms, but bioavailable nitrogen is often short in supply thus limiting primary production. The largest input of nitrogen to the marine environment is by N2-fixation, the transformation of inert N2 gas into bioavailable ammonium by a distinct group of microbes. Hence, N2-fixation bypasses nitrogen limitation and stimulates productivity in oligotrophic regions of the marine biosphere.

Extensive blooms of N2-fixing cyanobacteria occur regularly during summer in the Baltic Sea. N2-fixation during these blooms adds several hundred kilotons of new nitrogen into the Baltic Proper, which is similar in magnitude to the annual nitrogen load by riverine discharge and more than twice the atmospheric nitrogen deposition in this area. N2-fixing cyanobacteria are therefore a critical constituent of nitrogen cycling in the Baltic Sea. In this thesis N2 fixation of common cyanobacteria in the Baltic Sea and the direct fate of newly fixed nitrogen in otherwise nitrogen-impoverished waters were investigated. Initially, the commonly used 15N-stable isotope assay for N2-fixation measurements was evaluated and optimized in terms of reliability and practicality (Paper I), and later applied for N2-fixation assessments (Paper II–IV). N2 fixation in surface waters of the Baltic Sea was restricted to large filamentous heterocystous cyanobacteria (Aphanizomenon sp., Nodularia spumigena, Dolichospermum spp.) and absent in smaller filamentous cyanobacteria such as Pseudanabaena sp., and unicellular and colonial picocyanobacteria (Paper II-III). Most of the N2-fixation in the Northern Baltic Proper was contributed by Aphanizomenon sp. due to its high abundance throughout the summer and similar rates of specific N2-fixation as Dolichospermum spp. and N. spumigena. Specific N2 fixation was substantially higher near the coast than in an offshore region (Paper II). Half of the fixed nitrogen was released as ammonium at the site near the coast and taken up by non-N2-fixing organisms including phototrophic and heterotrophic, prokaryotic and eukaryotic planktonic organisms. Newly fixed nitrogen was thereby rapidly turned-over in the nitrogen-depleted waters (Paper III). In colonies of N. spumigena even the potential for a complete nitrogen cycle condensed to a microcosm of a few millimeters could be demonstrated (Paper IV). Cyanobacterial colonies can therefore be hot-spots of nitrogen transformation processes potentially including nitrogen gain, recycling and loss processes. In conclusion, blooms of cyanobacteria are instrumental for productivity and CO2 sequestration in the Baltic Sea. These findings advance our understanding of biogeochemical cycles and ecosystem functioning in relation to cyanobacterial blooms in the Baltic Sea with relevance for both ecosystem-based management in the Baltic Sea, and N2-fixation and nitrogen cycling in the global ocean.

Place, publisher, year, edition, pages
Stockholm: Department of Ecology, Environment and Plant Sciences, Stockholm University, 2015. 41 p.
Keyword
biogeochemistry, nitrogen cycling, nitrogen fixation, cyanobacteria, Baltic Sea
National Category
Ecology
Research subject
Marine Ecology
Identifiers
urn:nbn:se:su:diva-122080 (URN)978-91-7649-278-9 (ISBN)
Public defence
2015-11-27, sal P216, NPQ-huset, Svante Arrhenius väg 20 A, Stockholm, 13:00 (English)
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Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Available from: 2015-11-05 Created: 2015-10-23 Last updated: 2015-12-15Bibliographically approved

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