KEOPS planning meeting 2 –3 March 2004, Marseille

1. KEOPS planning meeting 2 –3 March 2004, Marseille Contribution from Royal NIOZ trace metal/phytoplankton group Klaas Timmermans (presenting) Marcel Veldhuis Corina Brussaard Patrick Laan Loes Gerringa Hein de Baar Focus on trace metals (iron) and interactions with phytoplankton (diatoms) THIS PRESENTATION: I) Analytical chemistry II) Phytoplankton / viruses Royal Netherlands Institute for Sea Research, Texel, The Netherlands You are free to use (parts of) this presentation, but notify the presenting author.

2. But first, why do we want to participate in KEOPS ??? • Research interest in: • Iron distribution (horizontal, vertical) • speciation (redox, organic complexation) • sources (sediment, dust) • interaction with • Phytoplankton (physiology, distribution) + phytoplankton viruses • Extension of Southern Ocean work, natural Fe enrichment. • All of which should lead to better insight in Si, N, P cycles, • the role of phytoplankton as forcing factors, and the interaction • with climate change.

3. I). ANALYTICAL CHEMISTRY (focus on iron). DISTRIBUTION : Surface sampling (torpedo). Depth profiles upto 4000 m (winch + clean CTD frame + GoFlo’s). SPECIATION: Total dissolved Fe, Fe3+ , Fe2+ : FIA-CL Organic complexation of Fe: voltammetry SOURCES: IRONAGES project 2000 – 2003. WP 1 :IRON from below: sediment as sources of Fe (Gulf of Biscay). WP2: IRON form above: Canary Basin (dust)

4. tubing to the ship tube inlet Surface sampling: ‘non-iron’ fish Trace metal clean sampling Filtration Tube into clean container

5. The usual suspects inside the NIOZ clean laboratory van………

6. Alternative for GoFlo’s on the wire: NIOZ KLEY FRANCE deep sea winch 7000m kevlar cable (16 mm) with internal signal cables, proven to be CLEAN.

7. NIOZ Epoxy-coated stainless steelframe; 22 trace metal clean sampling bottles : NOEX or GloFlo.

8. driver unit pneumatics Air tubes link GoFlo with rotating ball valves NOEX expanding silicone closures

9. Routine deep profiling with ultraclean CTD frame and cable: GOFlo on CTD-frame GOFlo on single wire Courtesy: Geraldine Sarthou, Stephane Blain, Patrick Laan, Klaas Timmermans October 2003 cruise IRONAGES-3 off West Africa

10. How can we be of additional value for the KEOPS cruises ? (Analytical chemistry) KEOPS OBJECTIVE 1. Mechanisms of natural iron fertilisation c) Iron distribution and speciation. “low” and “high” iron sites & transects: iron clean sampling and analysis. For surface sampling: towed fish For depth profiles: KLEY FRANCE winch with CTD rosette and GoFlo’s or GoFlo’s mounted on Kevlar wire. Sample treatment/analysis inside clean laboratory van. Dissolved Fe + organic complexation of Fe (shipboard)

11. II. Phytoplankton/viruses BIOGEOCHEMISTRY : studies at circulation of chemical elements in the ocean, between ocean and atmosphere, as governed by anabolic and catabolic activities of living organisms. Primary producers are main drivers of biogeochemical cycles of C, N, Si and P. Main functional groups: diatoms, coccolithophorids, Phaeocystis , picophytoplankton. In HNLC area’s, the large diatoms show the stongest response to Fe fertilisation and nutrient uptake is under strong influence of availability of Fe.

12. Specific research questions. Phytoplankton physiology: - Co-limitation of diatoms by iron and silicate, iron and light. - Elemental composition of diatoms in relation to iron, silicate. - Size differential effect of Fe fertilisation. - Optimalisation of biogeochemical models. Phytoplankton viruses: - Role of viruses in controlling small phytoplankton species. - Role of viruses in nutrient recycling.

13. And in practice: Phytoplankton distribution: abundance and species compostion of natural assemblages. Phytoplankton physiology: growth/mortality, co-limitation, nutrient uptake in relation to iron (and silicate and light) of natural assemblages and single species. Phytoplankton viruses: abundance, diversity and lysis rates. Laboratory experiments with the “bloom forming diatom species” Natural filtered Southern Ocean water: High Nutrient, Low Chlorophyl (HNLC) waters, no additions other than iron (as FeCl3 or dust) What is the effect of iron on: µmax and Km? nutrient (N, P, Si) uptake (ratios) ?

14. Klaas never travels alone... single species cultures of large Southern Ocean diatoms) Thalassiosira sp. Actinocyclus sp. 80 µm 140 µm 80 µm Fragilariopsis kerguelensis 70 µm Chaetoceros dichaeta

15. Growth rate (d-1) Shipboard experiments ambient dissolved Fe 0 0 . . 6 6 C . b r e v i s C . d i c h a e t a 0 0 . . 5 5 0 0 . . 4 4 KmC.brevis 0.59 x 10-12 M 0 0 . . 3 3 KmC.dichaeta: 1.12 x 10-9 M 0 0 . . 2 2 0 0 . . 1 1 0 0 . . 0 0 F e d i s s o l v e d ( M ) - - 0 0 . . 1 1 - 1 4 - 1 3 - 1 2 - 1 1 - 1 0 - 9 - 8 1 0 1 0 1 0 1 0 1 0 1 0 1 0 In the Southern Ocean: Large C. dichaeta is mostly Fe-limited except after Fe supply. Small C. brevis is never Fe-limited but grazer-controlled (viruses ???). - 1 4 - 1 3 - 1 2 - 1 1 - 1 0 - 9 - 8 1 0 1 0 1 0 1 0 1 0 1 0 1 0 Timmermans et al. Limnol. Oceanogr, 2001.

16. Growthrates versus [Fediss] Experiments in HNLC Southern Ocean waters, nothing added but Fe Fragilariopsis kerguelensis Growth rate (d-1) • µmax Km • (d-1) Fediss (nM) • Actinocyclus sp. 0.34 0.98 • Thalassiosira sp. 0.31 0.62 • C. pennatum 0.36 0.57 • F. kerguelensis 0.39 0.19 Fediss (nM) Laboratory experiments ambient [Fediss]; 0.2 nM Timmermans et al. Limnol Oceanogr. (almost accepted)

17. Si uptake: increases under Fe limitation N uptake: decreases under Fe limitation P uptake: variable effects of Fe limitation Nutrient uptake versus [Fediss] Effect on molar uptake ratios Molar uptake ratio N:P = 16 Fragilariopsis kerguelensis Si:N = 7.5 N:P = 5.8 Actinocyclus sp. Si:N = 2 Laboratory experiments Fediss (nM)

18. Where does the iron come from, what is bioavailable ?combined physico-chemical and biological approach (diatoms as indicators of availability of Fe originating from dust).

19. Physico-chemical analyses of the dust DUST(only fraction <63 µm used): Namibia : silty clay loam Mauretania: sand Characterisation: 1) Specific surface area 2) Mineral composition 3) Fe: Amorphous Fe Crystalline Fe Total Fe 4) Dissolution of particles in seawater / concentration of Fediss All indicating Namibia > Mauretania , BUT is there a link with bioavailability of Fe from the dust ???

20. “if you cannot measure it chemically, let the diatoms tell it !!” Growth rate (d-1) Actinocyclus sp. 1 Growth curve (FeCl3) known amount of dust gives growth rate 2 3 Fediss (nM) “bioavailable” Fe from dust

21. Growth responses Additions: = 1 mg Maur., = 5 mg Maur., = 1 mgNam. = 5 mg Nam. FeCL3 additions dust addition: growth rates increase Namibia > Mauretania

22. Overall RESULTS Addition [Fediss] (nM) Actinocyclus sp. Thalassiosira sp. DUST measured “says” “says” 1 mg Namibia 12.8 > 5.0 nM (?%) 0.12 nM (1%) 5 mg Namibia 44.2 > 5.0 nM (?%) 0.32 nM (1%) 1 mg Mauretania 1.7 0.50 nM (29%) 0.04 nM (2%) 5 mg Mauretania 5.8 0.36 nM (6%) 0.04 nM (1%) CONCLUSION. Yes, dust is a source of iron for the ocean, but only a (small) part of the iron that dissolves from the dust is bioavailable !! Visser, Gerringa, van der Gaast, de Baar andTimmermans (2003). J. Phycol.

23. How can we be of additional value for the KEOPS cruises ? (phytoplankton / viruses) KEOPS OBJECTIVE 2.1 aerosols as source of Fe to the ocean. Use of phytoplankton (natural assemblages, single diatom species) as bio-indicators of Fe originating from aerosol dust. KEOPS OBJECTIVE 3. Factors regulating phytoplankton growth and species composition, structure of the phytoplankton communities, etc. In situ : Responses of phytoplankton (diatoms) along KEOPS gradients and on “low” and “high” iron sites. How ? Quantification and probing of natural phytoplankton and virus assemblages.

24. Main approach for on board experiments (OBEX): Culturing of and experimenting with Antarctic phytoplankton in natural seawater, as indicators of bioavailable iron (sediments or dust), silicate and/or light. Viral induced mortality of phytoplankton in “low” and “high” iron sites. How ? Incubations with single species SO diatoms (large & small). OBEX 1. Incubations with natural (diatom) phytoplankton population: translocation experiments from Fe-rich, Si-poor to Fe –poor and Si-rich waters (and vice versa). OBEX 1. Fe and Si recycling due to physiological automortality, virus mediated cell death or micro-zooplankton grazing of the phytoplankton. OBEX 2 and 3.

25. Parameters: Growth and mortality of phytoplankton (diatoms): Physiological automortality with SYTOX viability assay. Next slide Silification PDMPO probe (Marie Curie fellowship application , Karine Leblanc) Nutrient uptake  nutrient uptake ratios Photosynthetic efficiency (Fv/Fm) Virus mediated cell death or micro-zooplankton grazing “EQUIPMENT”. Flowcytometry: 2 x Coulter + BD Pulse Amplitude Modulated fluorometer (PAM)  Fv/Fm Microscopy Large Antarctic diatoms: Actinocyclus, Thalassiosira , Eucampia, Proboscia. (more new –fresh- requested from EIFEX cruise) Small Antarctic diatoms: Chaetoceros brevis, Thalassiosira antarctica !!!!! ALL TRACE METAL CLEAN !!!!!

26. growth rate (d-1) T. antarctica Diatom automortality SILICATE SYTOX viability probe for individual cells, quantified using flowcytometry. growth rate lysis rate (d-1) Lysis rates increase with low silicate concentrations. Si (µM) lysis rate Si (µM)

27. Summarising for the KEOPS in situ & OBEX 1 - 3: Water from “low” and “high” iron sites (and varying silicate) + light Natural phytoplankton and single species responses of growth nutrient uptake (ratios) auto mortality viral induced mortality silification photosynthetic efficiency

28. Jan. 1999 Jan. 2000 Jan. 2001 And additionally, proposal submitted for Real time satellite imagery of SEAWIFFS + interpretation ? Jan. 2005

29. All of which should lead to better insight in Si, N, P (+ C) cycles, the role of phytoplankton as forcing factors, and the interaction with climate change. At the end we hope to say: “ …… still confused, but now on a much higher level ..…”.