What nutrients are controlled ? Who’s doing the controlling ? Weekly Theme: The Ocean as a Microbial Habitat Daily Theme: Ocean biogeochemical variability M J Perry Lecture: Biological control of ocean nutrients C-MORE, 2 June 2010 What you should know by the end of lecture (at least something about): What nutrients are controlled ? Who’s doing the controlling ? Why are they doing it ? How do they do it ?

Gruber 2008

Upper ocean nutrient cycles Dissolved inorganic nutrients Uptake (Drawdown) Particulate organic nutrients Remineralization (Recycling) DON mixing sinking remineralization and denitrification at depth

Key biological transformations of elements (nutrients). Uptake of dissolved inorganic nutrients and assimilation into particulate organic nutrients – by autotrophs: phytoplankton and bacteria {little ‘b’ includes Archaea}; some heterotrophic bacterial uptake (e.g., NH4+), but won’t discuss today. Consumption of organic particles by heterotrophs – leads to synthesis of new organic compounds; still organic nutrient Excretion of dissolved inorganic and organic nutrients – as by-product of heterotrophic metabolism Removal of nutrient from system Sinking organic particles (long time to return), denitrification (permanent) 5) Change in bio-availability of nutrient – N2 fixation, chelation of trace metals

Not all elements in seawater are considered as ‘nutrients’. Major ions in seawater: NaCl constitutes 85.65% of total dissolved salts; together, six major ions make up 99% of dissolved salts. Cl -, Na+, Mg+2, SO4-2, Ca+2, K+ [mmol/kg range] The major ions are mostly conservative and are present in ~ constant proportions throughout world oceans. Neumann & Peirson 1966

Not all elements in cells are considered as ‘nutrients’. Macro elements in cells – relative % of cell biomass (in decreasing order; not including H2O): C, H, O, N, P, S, Mg, K, Ca and Si (for diatoms, radiolarians) Red elements are in low concentration in ocean (µmol/kg range), in contrast to major salts on previous page (mmol/kg). Micronutrients in cell – are minor % of mass, but important: Micronutrients are typically trace metals: Fe+3 and Fe+2 – nanoM or sub-nanoM range and other elements (Mn, Cu, Zn, Mo, Cd, V, Co, etc.) Trace metals –> metaloproteins (electron transfer, enzyme catalysis). Enzyme/cofactor requirements include Ferredoxin (Fe), carbonic anhydrase (Zn), other trace metals…..

Seawater – top 6 ions Cl - 546 mmol/kg Na+ 469 mmol/kg Mg+2 52.7 mmol/kg SO4-2 28 mmol/kg Ca+2 10.3 mmol/kg K+ 10.2 mmol/kg C (DIC) 2.2 mmol/kg NaCl constitutes 85.65% of total dissolved salts. These six major ions make up 99% of dissolved salts. Plankton – top elements C, H, O, N, P, S, Mg, K, Ca and Si; concentrations lower at surface: N < 40 mol/kg P < 3 mol/kg Si < 150 mol/kg Trace metals [<nmol/kg] Elements in red can be limiting nutrients – limiting both to phytoplankton biomass and growth rate.

Key biological transformations of elements (nutrients). I) Uptake of dissolved inorganic nutrients by autotrophs (phytoplankton, bacteria) leads to assimilation of nutrients into organic compounds. Increase in cell biomass leads to synthesis of new cells. Drawdown of nutrient is flip side of uptake. Waniek. 2003. J. Mar. Syst. 39:57 (spring bloom)

North Atlantic spring bloom observed from a float, shows nitrate drawdown and chlorophyll increase from mid April to late May 2008. Chlorophyll Nitrate Bagniewski et al., in prep.

1a) Nutrient uptake has been described by Michaelis Menton enzyme kinetics. Paasche (1973) Vmax Si concentration V, Si uptake rate S0 KS V = uptake rate (mole/cell/time Vmax = maximal rate S = concentration (mol/kg) KS = substrate concentration at 0.5 Vmax DIN Uptake rate ~ function of: *nutrient concentration at cell surface (DIN; related to cell size and relative motion), * number/types of transporter (dots), * internal concentration of nutrient (N), and * growth rate (metabolic need). DIN DIN DIN N DIN DIN DIN DIN DIN Parameters vary w/ size, species, methodology.

Nutrient acquisition is affected by size: concentration vs. distance from cell surface. Steeper gradient for small cells - diffusion alone not so bad (PS bacteria)

Swimming or sinking improves flux; as does other interactions with flow field

Nutrient could be N, P, Si, Fe, Zn, etc. 1b) Nutrient concentration can regulate growth rate (shape of curve similar to uptake kinetics; KS for growth). Growth rate Nutrient concentration Culture data of Sommer Nutrient could be N, P, Si, Fe, Zn, etc.

Nutrient concentration can regulate growth rate, but real world relationships rarely looks like cartoon. Growth rate Nutrient concentration Goericke (2002) Limnol. Oceanogr., 47: 1307 Nitrate and growth rate of specific phytoplantkon in Arabian Sea.

Nutrient concentration does not tell the whole story. Ambient concentrations of nitrate do not reflect nutrient flux (from mixing, diffusion or recycling through heterotroph excretion). At low concentrations growth rate may be limited –– but if flux is large enough, growth rate may NOT be nutrient limited. Goericke (2002) Limnol. Oceanogr., 47: 1307

growth rate (doublings d- -1) 1c) Internal nutrient concentrations may be a better predictor of growth rate (also note that shape of curve similar to uptake kinetics). Problem -- internal concentrations are difficult to measure growth rate (doublings d- -1) 10 -12 g N per cell Mimimal requirement for structural cell components Valiela, 1995; data of Goldman and McCarthy, 1978)

But . . . new single-cell techniques help quantify internal concentrations for some elements. diatoms picophyto heteroflag. False-color X-ray fluorescence of Fe in silicoflagellate – Southern Ocean Twining et al. 2008. J. Eukaryot. Microbiol. 55: 155

Single-cell element data from preceding figure. Units are µmole / liter cell volume Twining et al. 2004. Limnol. Oceanogr. 49: 2115

Nutrient could be N, P, Si, Fe, Zn, etc. 1d) Nutrient concentration sets upper limit to biomass. El Nino Phytoplankton biomass Nutrient concentration Nutrient could be N, P, Si, Fe, Zn, etc. La Nina

Chlorophyll (~ biomass) vs. DIN concentration The actual biomass produced may be different – if other nutrients are at limiting concentrations or if environmental conditions are suboptimal (Fe - for example, HNLC regions, light, temperature, ). Nutrient concentration Phytoplankton biomass Chlorophyll (~ biomass) vs. DIN concentration http://www.ozcoasts.org.au/indicators/water_column_nutrients.jsp

D’Asaro et al. – Lagranian float in 2008 North Atlantic Bloom The accumulated biomass may be less than observed – due to loss after production (grazing, sinking, conversion to dissolved organics, ?) Nitrate drawdown converted to POC (Redfield) Beam attenuation converted to POC D’Asaro et al. – Lagranian float in 2008 North Atlantic Bloom

1e) Can one predict drawdown of one nutrient from drawdown in another? Nitrate Carbon

autotrophy <–> heterotrophy connection Redfield ratio (1963) – empirical/statistical relationship of molar average elemental ratios Alfred Redfield Biological Redfield ratio C: N: P ~ 106: 16: 1 average molar elemental composition of particles (organisms) Geochemical Redfield ratio C: N: P ~ 105: 15: 1 deep-water ions: nitrate, phosphate, and non-calcite DIC autotrophy <–> heterotrophy connection (autotrophy creates organics, respiration remineralizes nutrients)

The biological Redfield ratio has taxonomic variability. C:P N:P C:N The biological Redfield ratio has taxonomic variability. Co-evolution of organisms & ocean chemistry C:N:P composition varies between phyla and superfamilies. Phytoplankton C:P, N:P and C:N (mol:mol) ratios are grouped phylogenetically – Prasinophyceae (Prasino) and Chlorophyceae (Chloro) are members of the green (G) plastid superfamily whereas Dinophyceae (Dino), Prymnesiophyceae (Prymn) and Bacillariophyceae (Diatoms) are members of the red (R) plastid superfamily. Error bars indicate standard errors. Quigg et al. 2003. Nature 425:291

Redfield ratio has physiological variability. Structural cell components see figure below Growth- related N:P P-rich RNA  low N:P ratio Nutrient acquisition N:P N-rich enzymes high N:P ratio Storage N:P P can be stored if N is limiting Structural N:P ratio of 29 species of freshwater and marine phytoplankton. Redfield ratio is shown, as is the theoretical range predicted by the model under 3 cases: exponential growth (Opt exp), competitive equilibrium with light, N- and P- limitation. 0 48 Structural N:P ratio Klausmeier et al. 2004 Nature 429: 171

Redfield ratio is changed by nutrient limitation. C:P N:P Growth rate, d-1 1,600 120 For a cell limited by P, relative concentration of other elements in the cell will increase: When P is not limiting (far right), growth rates can be maximal. When P is limiting (left), growth rates are lower.

Redfield did not derive a ratio for trace metals, but others have Redfield did not derive a ratio for trace metals, but others have. (Used in estimating Fe-fertilization carbon credits). Stoichiometry shows (Ho et al, 2003, J. Phycol. 39: 1145) taxonomic patterns related to evolution, greater variability for metals that substitute in metaloenzymes (e.g., Zn, Co, Cd), reflects biogeography (coastal vs. oceanic). Others studies show some phytoplankton can synthesize non-metalo co-factors under Fe limitation or store Fe (ferritin) after an Fe pulse.

Ho et al, 2003, J. Phycol. 39: 1145

http://www. awi. de/fileadmin/user_upload/News/Press_Releases/2004/1 http://www.awi.de/fileadmin/user_upload/News/Press_Releases/2004/1._Quarter/Thalassiosira_w.jpg Diatoms have a unique requirement for Si (as do radiolarians). Ratios of diatom Si to other elements vary. Average Si: N ratio of 27 species = 1.05. But >10-fold variation (p.3 to 4) independent of variable growth conditions or nutrient availability. (Brzezinski, 1985, cited in Marchetti & Cassar, 2009. Geobiology 7: 419 Fe-limited diatoms have thicker frustules (sink faster; more resistant to dissolution; what’s impact on Si cycle? Si limiting Si not limiting Martin-Jézéquel et al. 2002 J. Phycol. 36: 821

Key biological transformations of elements (nutrients). Uptake of dissolved inorganic nutrients and assimilation into particulate organic nutrients – by autotrophs: phytoplankton and bacteria {little ‘b’ includes Archaea}; some heterotrophic bacterial uptake (e.g., NH4+), but won’t discuss today. Consumption of organic particles by heterotrophs – leads to synthesis of new organic compounds; still organic nutrient Excretion of dissolved inorganic and organic nutrients – as by-product of heterotrophic metabolism (some organic excretion by autotrophs under certain circumstances) Removal of nutrient from system Sinking organic particles (long time to return), denitrification (permanent) 5) Change in bio-availability of nutrient – N2 fixation, chelation of trace metals

From a heterotroph’s perspective, organic particle consumption leads to growth and reproduction. But consumption is not efficient. Nutrients are released – particulate organic, dissolved organic, and dissolved inorganic. Consumption Defecation & sloppy feeding Assimilation Respiration (energy for maintenance, biosynthesis, activity, etc.) Growth & Reproduction Molt PON DIN and DON

The more trophic transfers, the more remineralization.

In the Microbial Loop, it’s almost all trophic transfers, so recycling of nutrients is efficient. (Net production of new POC is not efficient.) lysis DOC Picoautotrophs DIN Microbial Loop: DOC POC DIC DIN PON DIN HNF and ciliates have v. high respiration rates

Where are the gellies?

Heterotrophic Prokaryotes Heterotrophic Prokaryotes * Sink for dissolved organics (organics excreted by autotrophs, products of viral lysis, inefficient zooplankton feeding, prokaryotic exoenzymes) * Sink or source of dissolved inorganic nutrients? [depends on the C:N:P ratio of dissolved organics] Heterotrophic Protists * Sink for particulate organics (ingest mostly small bacterial-sized particles; repackage them into larger particles) * Source of dissolved inorganic nutrients to phytoplankton AND bacteria (excretion or remineralization or regeneration of N, P, Fe, etc. by virtue of their very high metabolic rates) * Enhancement of food quality for mesozooplankton

How much C, N, etc. are remineralized. Are bacteria remineralizers How much C, N, etc. are remineralized? Are bacteria remineralizers? or are they consumers of N ? (same analogy for P, Fe, etc.) Depends on the quality of DOC Low C/N; low BGE more C -> CO2 little N recycled High C/N: high BGE more C -> biomass more N recycled Kirchman, Microbial Ecology of the Oceans

Key biological transformations of elements (nutrients). Uptake of dissolved inorganic nutrients and assimilation into particulate organic nutrients – by autotrophs: phytoplankton and bacteria {little ‘b’ includes Archaea}; some heterotrophic bacterial uptake (e.g., NH4+), but won’t discuss today. Consumption of organic particles by heterotrophs – leads to synthesis of new organic compounds; still organic nutrient Excretion of dissolved inorganic and organic nutrients – as by-product of heterotrophic metabolism (some organic excretion by autotrophs under certain circumstances) Removal of nutrient from system Sinking organic particles, vertically migrating zooplankton, denitrification 5) Change in bio-availability of nutrient – N2 fixation, chelation of trace metals

Sinking organic material (zooplankton fecal pellets, dead zooplankton and fish, phytoplankton aggregates) removes nutrients from the euphotic zone. WHOI sediment trap collections http://www.whoi.edu/oceanus/viewArticle.do?id=2372 Particle aggregation and sinking. Burd & Jackson, 2009. Ann. Rev. Mar. Sc.

Organic material is consumed and nutrients are remineralized if particles slowly sink. Subsurface oxygen minimum is a signature of respiration. Oxygen (µmol/kg) Martin et al. 1987 Johnson, http://www.mbari.org/chemsensor/pteo.htm

Diel and seasonal migration of zooplankton can transport nutrients out of euphotic zone. http://www.sintef.no/project/calanus/graphics/calanus.jpg Longhurst and Harrison. 1988. Vertical nitrogen flux from the oceanic photic zone by diel migrant zooplankton and nekton. Deep-Sea Res 35: 881.

Polyphosphate synthesis by diatoms may play a role in permanent removal of P from the recycling pool. (Organic phosphonates may sequester P for specific groups.) Phosphonate use by Trichodesmium (Dave Karl lecture) In sediment, diatom polyphosphate enables transition to apatite (crystalline phosphorus calcium deposit) http://www.bone.pentax.jp/newceramics_e.php Diaz et al. 2008. Science 320: 652

Denitrification removes bio-available from the ocean. (and decreases the Redfield N:P ratio.) Net effect is removal of available N from ocean in anaerobic conditions: Denitrification – when O2 is low, some bacteria can use NO3- as a terminal electron acceptor (use nitrate as a substitute for oxygen). Heterotrophic. NO3− → NO2− → NO + N2O → N2 Anammox (ANaerobic AMMonium Oxidation). Autotrophic. NH4+ + NO2− → N2 + 2H2O There is more residual PO4-3, so N:P ratio decreases. Tyrrell, 2001

Key biological transformations of elements (nutrients). Uptake of dissolved inorganic nutrients and assimilation into particulate organic nutrients – by autotrophs: phytoplankton and bacteria {little ‘b’ includes Archaea}; some heterotrophic bacterial uptake (e.g., NH4+), but won’t discuss today. Consumption of organic particles by heterotrophs – leads to synthesis of new organic compounds; still organic nutrient Excretion of dissolved inorganic and organic nutrients – as by-product of heterotrophic metabolism (some organic excretion by autotrophs under certain circumstances) Removal of nutrient from system Sinking organic particles, vertically migrating zooplankton, denitrification 5) Change in bio-availability of nutrient – N2 fixation, chelation of trace metals

Trace metals are complexed by different classes of organic ligands Trace metals are complexed by different classes of organic ligands. Different ligands appear to give selective advantage to different taxanomic groups. Stronger class of chelator. Include siderophores. Cyanobacteria are able to access trace metal. Eukaryotes are less able. Weaker class of chelator. Thought to be detrital in nature. Metal is more accessible to eukaryotes. Hutchins et al. 1999 . Nature 400, 858

N2 fixation adds bio-available N to the ocean N2 fixation adds bio-available N to the ocean. (and increases the Redfield N:P ratio). N2 + 6 H+ + 6 e− → 2 NH3 More Fe input to Sargasso Sea –> more N2 fixation –> higher N:P ratios. Wu, Sunda, Boyle, Karl (2000. Science 289: 759) suggest Aeolian Fe (increases N through N2 fixation. (There is also a shift to N2-fixing cyanobacterial species.) Redfield ratio BATS vs. HOTS

Is the Redfield ratio still useful? (it does vary) Yes – some predictive power (e.g., ~ C fixed per N; eutrophication) Yes – insight into autotrophy <–> heterotrophy connection (elements are incorporated into organics and released or remineralized) Yes – sight into opposing biological processes in nitrogen cycle: N2 fixation adds N and denitrification removes N from ocean

Key biological transformations of elements (nutrients). Uptake of dissolved inorganic nutrients and assimilation into particulate organic nutrients – by autotrophs: phytoplankton and bacteria {little ‘b’ includes Archaea}; some heterotrophic bacterial uptake (e.g., NH4+), but won’t discuss today. Consumption of organic particles by heterotrophs – leads to synthesis of new organic compounds; still organic nutrient Excretion of dissolved inorganic and organic nutrients – as by-product of heterotrophic metabolism Removal of nutrient from system Sinking organic particles (long time to return), denitrification (permanent) 5) Change in bio-availability of nutrient – N2 fixation, chelation of trace metals