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Final Report Summary - DIADOM (Interactions between nitrogen-fixers (diazotrophs) and dissolved organic matter in the ocean)

The goal of the DIADOM project was to investigate how the biological fixation of dinitrogen (N2) interacts with organic matter in marine systems. Biological N2 fixation is a process performed by prokaryote microbes (called ‘diazotrophs’) in marine systems that provides bioavailable nutrients, thus fueling primary production and consequent carbon dioxide uptake. In summary, N2 fixation provides the resources needed by the ocean biota to alleviate global warming.
Biological N2 fixation had been long attributed solely to autotrophic (photosynthetic) microbes belonging to the cyanobacteria, but more recent studies demonstrated that heterotrophic non-photosynthetic bacteria and archaea are more abundant and widespread in the oceans than their autotrophic counterparts. This discovery posed new questions and prompted new approaches to study oceanic N2 fixation: heterotrophic microbes are constrained by different environmental factors than autotrophic ones, thus significant changes in our conceptual and technical approaches was necessary. More specifically, heterotrophic microbes depend on organic matter availability for their nutrition and are a priori not constrained by the availability of light. Our hypothesis was that the activity and diversity of heterotrophic diazotrophs was controlled by organic matter composition and dynamics, and that their presence and activity could be important even below the sunlit upper layer of the ocean (i.e. >200 m).
To prove this hypothesis in the DIADOM project we have applied a unique coupling of organic matter geochemistry and molecular biology techniques, complemented by traditional microbial ecology approaches. The sampling has been performed during 3 cruises in the Southwest Pacific Ocean:
1) MoorSPICE cruise, 01/03-31/03/2014, onboard the R/V Thomas G. Thompson (USA)
2) OUTPACE cruise, 19/02-02/04/2015, onboard the R/V L’Atalante (France)
3) LAGON cruise, 13/01/2016, onboard the R/V Archamia (New Caledonia)

During these cruises we have:
• Mapped aphotic N2 fixation rates (mesopelagic depth range 200-1000 m), diazotroph abundance and diversity, and the chemical composition of organic matter, with the aim of finding spatial distribution patterns among these parameters
• Exposed natural photic and aphotic diazotroph populations to known sources of organic matter to investigate their responses in terms of activity and gene expression
• Performed experiments to isolate the heterotrophic diazotrophy signal from the autotrophic one

This work was complemented by the collaboration with two other projects in the Mediterranean Sea and the New Caledonian lagoon (Southwest Pacific), where we had the change to take further samples to test our hypothesis on diazotroph-organic matter interactions:
4) HOTMIX cruise, 01/04-30/05/2014, onboard the R/V Sarmiento de Gamboa (Spain)
5) Project DIADOM-Corail, which was performed thanks to several one-day surveys onboard the R/V Archamia (New Caledonia)

Samples from the HOTMIX cruise were provided by the team of Prof. Javier Arístegui (Instituto de Oceanografía y Cambio Global, Spain) and analyzed at MIO. For this collaboration we undertook a similar approach to that followed during the MoorSPICE cruise and studied patterns of aphotic N2 fixation related to organic matter in the deep Mediterranean Sea. The collaboration with the project DIADOM-Corail allowed us to study how the organic matter produced by corals (mucus) affects N2 fixation in the waters of the New Caledonian lagoon. Altogether, the collaboration with these projects has given us the opportunity to further test our hypothesis in environments governed by different environmental factors and ecological processes, strengthening our results and conclusions.
During the MoorSPICE cruise we measured N2 fixation in the mesopelagic layer of the Bismarck and Solomon Seas in the Southwest Pacific together with organic matter composition and the diversity of nifH genes (encoding for a subunit of the enzyme responsible for N2 fixation). Mesopelagic N2 fixation rates were significant (up to ~1 nmol N L-1 d-1) and comparable to those usually measured in open ocean waters. These rates were higher in the Bismarck Sea than in the Solomon Sea, and correlated with the higher availability of organic matter compounds in the former than in the latter. These differences in organic matter between the two areas likely reflect the higher primary production observed in the euphotic layer. The molecular analysis of nifH genes revealed a highly diverse diazotrophic community, suggesting that the N2 fixation rates measured in this study are accomplished by a variety of phylotypes at low abundances, rather than by a few phylotypes present at high abundances.
Besides exploring heterotrophic N2 fixation nutrition on organic matter, during the OUTPACE project we have explored the potential for a mixotrophic nutrition mode in natural colonies of Trichodesmium (the most important autotrophic diazotroph in the sea) inhabiting the euphotic zone. Mixotrophy (the ability to use both inorganic and organic nutrients) confers nutritional plasticity to microbes living in severely nutrient-limited environments, and changes fundamentally our understanding of marine planktonic foodwebs. Using nano-scale secondary ion mass spectrometry (nanoSIMS), we found an active uptake of 13C-labeled sugars and amino acids by Trichodesmium, and a significant enhancement of their N2 fixation rates, in the presence of conditions usually regarded as optimal for their development (high temperature, low nitrate concentrations). The mixotrophic nutrition of Trichodesmium likely has important implications for global nitrogen cycling models based on its N2 fixation activity as the main oceanic diazotroph.
The rest of the recent OUTPACE cruise results will be published later in 2016, and we are currently working on obtaining the data from the LAGON cruise. We highlight the logistic effort that oceanographic cruises and transfer of samples between laboratories in different countries take. The usual time lag between an oceanographic cruise and the publication of its results is 2-4 years, therefore we applied great efforts to reduce this lag significantly.
The results of the HOTMIX cruise depict as well a relationship between labile organic materials and aphotic N2 fixation in the Mediterranean Sea, though associated with different processes such as deep water formation events in the Mediterranean Sea. The results from the DIADOM-Corail project show that the organic mucus does not enhance planktonic N2 fixation, but nonetheless it is heavily colonized by heterotrophic diazotrophs, suggesting that the energetic requirements of planktonic and coral-colonizing diazotrophs are different.
Altogether, these findings reflect the importance of heterotrophic diazotrophy in the dark ocean, which remains unaccounted for in global estimates of the marine nitrogen budget -the main nutrient limiting primary production- and thus the ability of the ocean to uptake carbon dioxide and mitigate global warming. In summary, the results obtained during the DIADOM project demonstrate that heterotrophic N2 fixation is significant in the ocean and that it is related to the in situ available organic matter pool. These results significantly expand the areas of the ocean where N2 fixation was thought to occur and also indicate that not only heterotrophic but also autotrophic diazotrophs are affected by organic matter, hence changing our perception of resource utilization by diazotrophs in the ocean. These results impact the scientific community by changing former paradigms and prompting a suite of new scientific questions like how should these fluxes be included in current global biogeochemical models, and how organic matter spatial and temporal dynamics affect heterotrophic N2 fixation. Finally, our unique double approach combining organic geochemistry and molecular biology is pioneering in the field (to the best of our knowledge, less than 10 studies have included such an approach so far), and build upon current efforts to reconcile scientific disciplines in the field of microbial oceanography to allow opening the ‘black box’: which microbes use which substrates?

Exploitation and dissemination of results:
• 1 paper published in the PLOS ONE Journal
• 1 communication in the ASLO meeting 2015
• 4 papers submitted or in revision
• 4 papers in preparation

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