1. “Basic to the understanding of any ecosystem is knowledge of its food web, through which energy and materials flow. If microorganisms are major consumers in the sea, we need to know what kinds are the metabolically important ones and how they fit into the food web.” - Lawrence Pomeroy (Bioscience, 1974) “I presume that the numerous lower pelagic animals persist on the infusoria, which are known to abound in the open ocean: but on what, in the clear blue water, do these infusoria subsist?” - Charles Darwin (1845) Dissolved Organic Matter (DOM) and the Microbial “loop” CO 2 DOM CO 2 2015 CMORE Agouron Repeta Lecture 21

2. Cycling and composition of marine dissolved organic matter: A primer 1)What is the global distribution of dissolved organic matter? 1) How do we approach an understanding of its cycling and reactivity? 2)What techniques are used to characterize DOM composition? 1)Can we link composition to source and putative sinks? 2015 CMORE Agouron Repeta Lecture 22

3. Typical profile of dissolved organic carbon in the ocean Often measured as TOC Surface values typically 60-80 µM Deep water values @ 35-40 µM (implies some unknown feedback/ control of DOC values) Global inventory about 680 GT C Data from Peltzer and Hayward (1996) DSR TOC (µM) 2015 CMORE Agouron Repeta Lecture 23

4. Global circulation and the distribution of DOC Hansell et al. Oceanography 2009 2015 CMORE Agouron Repeta Lecture 24

5. 5 Stable carbon ( 13 C) isotopes suggest a marine source  13 C (per mil) Druffel et al., 1992 Carbon Dioxide Particulate Organic Carbon (POC) Dissolved Organic Carbon (DOC)

6. McNichol and Aluwihare 2007 2015 CMORE Agouron Repeta Lecture 26

7. Hansell et al. Oceanography 2009 (Inorganic carbon radiocarbon value) 2015 CMORE Agouron Repeta Lecture 27

8. DOC cycling via DO 14 C UV photooxidation Williams, Oeschger, and Kinney; Nature v224 (1969) 1880m -351 ‰ -3470+330 ybp 1920m -341 ‰ -3350+300 ybp Depth  14C(‰) Age 2015 CMORE Agouron Repeta Lecture 28

9. Radiocarbon in the Atlantic and Pacific Oceans Peter M. Williams and Ellen Druffel; Nature 1987, JGR 1992 Sargasso suspended POC Pacific suspended POC Pacific sinking POC 92015 CMORE Agouron Repeta Lecture 2

10. Radiocarbon in the Atlantic and Pacific Oceans DIC 14 C in surface waters of the Atlantic and Pacific have similar isotopic values. DOC is always older than DIC (by 2-3 kyrs in surface water) DIC POC DOC Sargasso suspended POC Pacific suspended POC Pacific sinking POC Sargasso suspended POC Pacific suspended POC Pacific sinking POC 102015 CMORE Agouron Repeta Lecture 2

11. Radiocarbon in the Atlantic and Pacific Oceans DIC 14 C in surface waters of the Atlantic and Pacific have similar isotopic values. DOC is always older than DIC (by 2-3 kyrs in surface water)  14 C of DIC and DOC is about the same in the deep Atlantic and Pacific Sargasso suspended POC Pacific suspended POC Pacific sinking POC Sargasso suspended POC Pacific suspended POC Pacific sinking POC 112015 CMORE Agouron Repeta Lecture 2

12. Radiocarbon based models of DOC cycling in the water column Atlantic surface water 14 C calc = -120 ‰ 14 C obs = -127 ‰ Pacific surface water 14 C calc = -147 ‰ 14 C obs = -148 ‰ [DOC] z (C14) z = [Deep](C14) D + [Z-Deep](C14) DIC DIC POC DOC 2015 CMORE Agouron Repeta Lecture 212

13. 2015 CMORE Agouron Repeta Lecture 213 Under this perspective, DOM is produced and rendered recalcitrant by marine microbes. combined with radiocarbon and deep sea DOC gradients, it suggests no or at best very slow removal of DOM in the deep sea

14. 2015 CMORE Agouron Repeta Lecture 214 Southern Ocean DOC-14 data suggests this model cannot be correct. Druffel and Bauer, 2000 GRL

15. 2015 CMORE Agouron Repeta Lecture 215 Depth (m) LIW 40  M 34  M Very rapid degradation of DOC in the Mediterranean Sea. If DOC is refractory, why does this happen? Santinelli et al. 2010. DSR C. Sanitelli et al. 2013

16. 2015 CMORE Agouron Repeta Lecture 216 Alternative models for DOC cycling in the deep sea

17. So what is DOC? 2015 CMORE Agouron Repeta Lecture 217

18. Salt Dissolved Organic Matter Composition – The Problem DOC 2015 CMORE Agouron Repeta Lecture 218

19. Separation based on size 1 nm pore @ 1 kD Selects for larger size (High Molecular Weight) fraction About 30-35% TOC (now up to 60% using electrically assisted UF) Membrane effects what is collected Cross or tangential flow filtration, Ultra- or nanofiltration 2015 CMORE Agouron Repeta Lecture 219

20. >1000 D DOM fraction < 1000 D DOM fraction Ultrafiltration of high molecular weight DOM (HMWDOM) 2015 CMORE Agouron Repeta Lecture 220

21. Seawater (filtered; pH = 2) Isolation of DOM by adsorption onto hydrophobic resins 10-20% of DOC Methanol or Ammonium hydroxide wash Selective chemical Adsorption 2015 CMORE Agouron Repeta Lecture 221

22. Can be tuned to different Nuclei of interest (C,N,P…). Gives information on functional groups which, combined with a knowledge of biochemicals can be used to deduce composition and origin. Nuclear Magnetic Resonance Spectroscopy (NMR) 2015 CMORE Agouron Repeta Lecture 222

23. 13 C Nuclear Magnetic Resonance Spectrum of high molecular weight dissolved organic matter (C/N = 15) Abundance Frequency 050100150200 2015 CMORE Agouron Repeta Lecture 223 O=C-O or O=CN H-C-OH O-C-O CH3

24. 13 CNMR of plankton tows Hedges et al GCA 2001 Sargasso Sea (surface) 13 CNMR of HMWDOM Chemical shift (ppm) 2015 CMORE Agouron Repeta Lecture 224

25. 3 m Sargasso Sea North Pacific Subtropical Gyre 13 CNMR spectra of HMWDOM 2015 CMORE Agouron Repeta Lecture 225

26. 3 m North Pacific Subtropical Gyre 13 CNMR spectra of HMWDOM 2015 CMORE Agouron Repeta Lecture 226 Lake Superior (USA) 3m

27. 2500 m 3 m 1800 m Sargasso SeaNorth Pacific HMWDOM in the deep sea Slide 5.17 2015 CMORE Agouron Repeta Lecture 227

28. 2015 CMORE Agouron Repeta Lecture 228

29. 2015 CMORE Agouron Repeta Lecture 229 Pedler et al. PNAS 2014 Degradation of DOM by marine microbes

30. 2015 CMORE Agouron Repeta Lecture 230 Sosa et al ISME 2015 Utilization of DOM polysaccharide by Marine microbes using dilution-to-extinction

31. Seawater (filtered; pH = 2) Isolation of DOM by adsorption onto hydrophobic resins 20-30% of DOC Methanol or Ammonium hydroxide wash Selective chemical Adsorption 2015 CMORE Agouron Repeta Lecture 231

32. http://antoine.frostburg.edu/chem/senese/101/glossary/m.shtml 2015 CMORE Agouron Repeta Lecture 232

33. 2015 CMORE Agouron Repeta Lecture 233

34. 2015 CMORE Agouron Repeta Lecture 234

35. Hertkorn Elemental Analysis of DOM in Weddell Sea Seawater 2015 CMORE Agouron Repeta Lecture 235

36. Surprisingly, there is a population of very low H/C and O/C organic matter Unusual molecular masses in DOM 2015 CMORE Agouron Repeta Lecture 236

37. 4.37 Source-specific studies Different responses to productivity levels in-situ SAR11 stays consistent Roseobacter and Bacteroidetes prefer high productivity Lability varies on a population scale Alonso-Saez et al AME 2007 Radiolabeled DOC derived from various phytoplankton sources Uptake of substrate by various heterotrophic groups shows connections between specific autotrophic & heterotrophic groups Sarmento et al EM 2012 2015 CMORE Agouron Repeta Lecture 2

38. 4.38 Cottrell & Kirchman AEM 2000 2015 CMORE Agouron Repeta Lecture 2

39. 4.39 Cottrell & Kirchman AEM 2000 2015 CMORE Agouron Repeta Lecture 2

40. CH 3 O HO CH 2 Aliphatic or “Humic” substances Concentration 40 µM Inventory = 640 GT C  14 C= -400 to -600‰ Annual flux = 0.1 GTC O -C-N - O O Non-reactive DOMSemi-reactive DOMVery reactive DOM Biopolymers (polysaccharides, proteins)* Concentration 0-40 µM Inventory = 10-20 GT C  14 C= modern (DIC) Annual flux = 10’s GT C? * = 80% of cell C, N Simple biomolecules (amino acids, sugars)* Concentration 1-2 µM Inventory = 0.1-0.3 GT C  14 C= modern (DIC) Annual flux = 10’s GT C? * = 10-20% of cell C, N Composition, reactivity, flux and distribution of DOM Slide 5.38 2015 CMORE Agouron Repeta Lecture 240

41. There is a large vertical gradient in DOC between surface and deep waters. Net production in the euphotic zone, net respiration in the mesopelagic zone. Stable carbon isotopes suggest most DOC originates from marine microbes, but this is not certain, as the isotopic values are open to interpretation. Radiocarbon measurements show that there is “new” DOC in the surface ocean, very “old” DOC at depth. Loss of about 30% of deep DOC between the North Atlantic and the North Pacific, along the path of deep water drift. Studies of chemical composition are handicapped with by our ability to sample DOM. DOM is sampled by two techniques, ultrafiltration which is based on the larger molecular size of some DOM relative to water and salt (high molecular weight DOM; HMWDOM) and solid phase extraction (SPE) that relies on chemical adsorption onto a hydrophobic surface. Lecture 2. Summary 2015 CMORE Agouron Repeta Lecture 241

42. Of the ~ 50-60% of DOM that can be recovered, and therefore characterized. Major techniques used to characterized DOM are nuclear magnetic resonance spectroscopy (NMR) and high resolution mass spectrometry (HR-MS). HMWDOM is largely “new” DOC and has a modern radiocarbon age when purified. HMWDOM is largely carbohydrate with a small amount of protein. HMWDOM carbohydrate has an unusual composition that has not been fully characterized. The source of the carbohydrate is not known. DOM extracted by adsorption onto a solid phase has an old radiocarbon age. So far this is very hard to analyze and characterize. It is clearly a complex mixture of organic matter with a significant amount of aliphatic character. The source of this DOM and the pathways that lead to its formation and removal are not known. Lecture 2. Summary 2015 CMORE Agouron Repeta Lecture 242

43. 2015 CMORE Agouron Repeta Lecture 243