Nitrogen fixation in tropical waters. Nutrition from the air? Julie LaRoche Cape Verde Observatory workshop: “Integrated, long term ocean-atmosphere observations in the tropical Atlantic” 8-10 January 07

Global Nitrogen Cycle Annual Fluxes (Units are Mt N y -1 ) Reservoirs in Mt N Oceans Combustion 22 Rivers 43 Burial <250 Denitrification N 2 -Fixation Lightning 3 Biomass 1200 N 2 -Fixation Denitrification 151 Denitrification 160 Haber-Bosch Process 93 Atmosphere 4.0 x 10 9 Sedimentary Rocks 1 x 10 9

Nitrogen Fixation N  N  organic-N compounds requires significant energy relatively few species can do the job: All are prokaryotes (photosynthetic cyanobacteria and heterotrophic bacteria) Requires the nitrogenase enzyme (23 iron atoms) North Atlantic sub-tropics and tropics seem to be regions of net N-fixation

Marine diazotrophs (nitrogen fixers): Some important questions Who is out there? How many are out there? Who is actively fixing nitrogen? How much nitrogen are they fixing? What factors control their growth? What is the importance of dust-supplied Fe? How will they adapt to future conditions?

Trichodesmium erythraeum IMS101 (6500 Kb) Fixes N 2 Trichodesmium erythraeum Trichodesmium photo: Courtesy of Pernilla Lundgren Genomes: Joint Genome Institute web site

M. barkerii Cyanobacteria α-proteobacteria 89 Cluster II 97 Cluster III 63 Cluster IV 99 γ,δ,ε, β -proteobacteria 60 Cluster I Recovered 354 nifH sequences 170 were unique Filamentous: Trichodesmium sp. 57% Unicellular: Unicellular A Cyanothece Synechocystis 32% 9% Heterotrophic 2% Anaerobic Global nifH Protein Tree (Langlois et al AEM)

Unicellular photoautrophic and heterotrophic diazotrophs are at best difficult to identify with traditional methods CyanobacteriaHeterotrophic bacteria AutofluorescenceDAPI staining / epifluorescence Crocosphaera watsonii WH8501 (4000 Kb)

Distribution and abundance of diazotrophs in the North Atlantic –Our approach: make a probe for each phylotype and use quantitative real-time PCR to count them (from the DNA) or see who is active (RNA sample). –Probes for 7 groups: Cyanobacteria: Filamentous, Croccosphaera, Cyanothece, unicellular A Heterotrophic bacterial phylotypes

Distribution and abundance of nifH phylotypes in the North Atlantic Trichodesmium-like  -proteobacteria AO Note the log scale Black dots are where we did not detect the group (Rebecca Langlois unpublished results)

a b c d e f g Snapshot of phylotype distribution Spatial variability between the groups Is this geographical or seasonal variability?

Distribution of cyanobacterial diazotrophs as a function of temperature Distribution of heterotrophic diazotrophs as a function of temperature

What controls dinitrogen fixation? 60L Control Initial Rates T=24 +P +Fe +NP + NFe + PFe + NPFe +Dust 1 +Dust 2 +N * +NO3 15 N 2 14 C Other Dust 1 Dust Saharan ss * 0.5mg L -1 Saharan ss * 2.0mgL -1 Saharan ss * 2.0 mg L -1 Saharan Atm † 2.0 mg L -1 * Saharan Soil (fine fraction) Collected in Southern Algeria † Atmospherically Collected Saharan Samples = 2 nM FeCl 3 +Fe = 0.2 µM NaH 2 PO 4 +P = 1 µM NH µM NO 3 - +N = 2 nM FeCl 3 +Fe = 0.2 µM NaH 2 PO 4 +P = 1 µM NH µM NO 3 - +N

Response of microbial community to nutrients and dust Combination of techniques: Molecular biology techniques Stable isotope Nutrient addition bioassays Flow cytometry Unicellular cyanobacteria (group A) at 5 m depth (nifH copy L -1 ) (Log scale)

Jickells T et al. (2005) Science, 308: 67 Study these effects where the dust input is large and variable BATS HOTS Cape Verde Compare to time series sites that are less affected by dust deposition Study these effects where the dust input is large and variable Cape Verde Compare to time series sites that are less affected by dust deposition Study these effects where the dust input is large and variable Cape Verde BATS HOTS Compare to time series sites that are less affected by dust deposition

Work planned at the Cape Verde time series site Up to now we have a snapshot view of the distribution of different diazotrophs in the North Atlantic Cape Verde site allows us to obtain a detailed view of diazotrophy and the role of dust in supplying iron Time series sampling: temporal and seasonal variations as a function of environmental factors such as dust deposition Intensive campains for bioassay experiments and diel cycle studies (one each year for the next three years)

Acknowledgements Past and present group members at the IFM-GEOMAR (Rebecca Langlois, Diana Hümmer, Matt Mills, Celine Ridame, Tania Klüver) Collaborators in the UK (Richard Geider, Margaret Davey, Mark Moore, Eric Achteberg, Angela Milne)