Approches moléculaires et isotopiques pour la caractérisation chimique de la matière organique dissoute (DOM)

Transcription

1 Approches moléculaires et isotopiques pour la caractérisation chimique de la matière organique dissoute (DOM) Christos Panagiotopoulos Mediterranean Institute of Oceanography (MIO) 11 Mai 2015

2 Talk outline. 1. State of the art on DOM chemical composition 2. Linking DOM chemical composition and radiocarbon 3. Compound specific radiocarbon analysis (CSRA)

3 Major Carbon Reservoirs Atmosphere 750 Gt C CO 2 Aeolian inputs Continental inputs Continental margins POC > 0.2 μm Bacteria Phyto Gt C DOC < 0.2 μm 700 Gt C Ocean Bacteria CO 2 DIC Gt C 0.1 Gt C Benthic layer release 6000 years Sédiment 90% Non-identified!! 150 Gt C t =?? an -1 Carlson and Hansell 2015; Repeta, 2015

4 How well do we know the composition of Organic matter? DOC Surface DOC 15-30% C characterized (AA-C + Lip-C +Sugar-C) Deep DOC < 10% C characterized (AA-C + Lip-C +Sugar-C) Most of the DOC remains uncharacterizable at the molecular level Benner (2002)

5 Natural Concentrations of compounds in DOM Carbohydrates: nmol/l for polysaccharides < 10 nmol/l for monomers Amino acids: similar range with carbohydrates Lipids: nmol/l Vs g/l of salt Measured concentrations quite close to the detection limits of the techniques employed Impossible to characterize the whole DOM with the current techniques

6 Conclusion: a. Very few profiles of these biochemicals published in literature for DOM b. Impossible to characterize the whole DOM with the current analytical techniques Do we have any alternatives? Get info from concentrated OM (i.e High Molecular Weight dissolved Organic matter HMWDOM)

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8 Approaches for the chemical characterization of DOM (1) Direct analyses (0.5-1 mg/l DOC) + - No contamination and artifacts Representative of DOM pool Low conc. of compounds (nmol) Salts (2) Analyses of concentrated DOM (ultrafiltration > 1kDa) + - Almost no salts 5-10 g of material Large vol. of samples required (>5000L) Only 25-30% DOC recovered NMR; MS-MS; D 14 C on individual comp. etc

9 flow in flow out DOM LMWDOM <1000 Da HMWDOM >1000 Da

10 HMWDOM collection Hawaii 3600 m Black sea, surf. Marseille 5m Rhône (Arles), surface Final product : % DOC; C/N = for seawater samples

11 absorbance HMWDOM composition by 13 CNMR C 6 H 12 O 6 CO H C 6 H 2 OH HO C H 4 C 5 O HO C H C 3 2 C 1 H HO H OH OCO OC 1 O OC 2-6 = 1/6 (hexoses) 1/5 (pentoses) COOH CH x ppm frequency Aluwihare et al., (1997); Repeta et al.(2002)

12 absorbance HMWDOM composition by 13 CNMR HMWDOM is mostly a mixture of carbohydrate like molecules [acyl polysaccharides (APS)]. HMWDOM comprize sugars (50-70%; OCO, HCOH groups), carboxylic + acetate (5%; COOH, CH 3 CO), alkyl (5%; CHx), proteins and humics (20%) HCOH However, after acid hydrolysis (HCl, TFA, H 2 SO 4 etc) only 15-20% of HMWDOM is recovered as neutral sugars. APS are present in both fresh and marine waters and their inventory is large (10-15 GTC at least) COOH OCO CH x ppm frequency Aluwihare et al., (1997); Repeta et al.(2002)

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14 Chemical composition of acyl polysaccharides (APS) Acides amines, lipides, Substances humiques, Carbone noir etc. HMW-DOM 50-70% % de HMW- DOM are acyl polysaccharides (APS) APS 10-15% are neutral sugars (fraction F4) 1.Hydrolysis 2.Purification of APS 13 C NMR of HMWDOM Liquid chromatography of APS HCOH F4 -CH3 F1 COOH OCO Acétamide 6-deoxy F2 F3 F ppm min

15 Detector response Detector response Chemical composition of the APS-F3 fraction by GC-MS OMe glucose F OMe fucose 3-OMe rhamnose 2-OMe galactose HO HO Time (min) 7 CH 2 CH 2 OH O CH 3 O OH Time (min) 3,6-dideoxyhexose; 2,3-di-O-methylrhmnose; 2,4-di-O-methylrhamnose; 2-O-methyl-pentose 2,6-di-O-methylhexose; 3,6-di-O-methylhexose; 2,3-di-O-methylhexose; 6-O-methylhexose Glucosamine, 2-O-methylheptoses

16 Results: Chemical structures found Panagiotopoulos et al Mar. Chem.

17 Results: Chemical structures found

18 Results 1. > 50 novel compounds (methylated, deoxy-, anhydro sugars) were identified in marine HMWDOM 2. These compounds account about 2-3% of the carbohydrate pool. 3. Although very few data are available in literature these methylated sugars have both bacterial and algal sources. However, more data are available for these compounds for bacterial lipopolysaccharides. 4. Anhydrosugars (i.e levoglucosan, mannosan, and galactosan) have been detected in DOM and their presence may suggest terrestrial burning biomass inputs.

19 Biogeochemical importance A. Methylated, dimethylated hexoses have been found in bacterial and algal cell walls as part of structural polysaccharides, however their chemical structure and function is poorly understood. Are these compounds contributors to the recalcitrant DOM? B. 3-6-dideoxysugars and heptoses have been found as antigenic polysaccharides in Gram-negative bacteria as well as in antibiotics. It is fundamental to get more info about bacteria structures. Do bacteria assimilate metylated or deoxysugars? C. Levoglucosan is a component of atmospheric smoke particles derived from wood burning (cellulose degradation product) and this is the FIRST time that has been found in DOM in tiny amounts. Does black carbon (part of the uncharacterizable DOM carbon) enter the ocean via atmospheric deposition? (this was assumed but not proven by molecular level analysis by Masiello & Druffel, 1998). Measurements of d 13 C of pure levoglucosan in surface and deeper DOM samples Possible collaboration with CEREGE??

20 Beyond Biochemistry Sugar moiety Sugar moiety Erythromycin Methylated 2,6 or 3,6 dideoxysugars Azythromycin ANTIOBIOTICS: Antibacterial agents The sugars moieties in antiobiotics actively contribute as recognition elements to the mechanism of action of the respective drug and their removal often results in the loss of all biological activity. IDENTIFY new sugar compounds in the HMWDOM may help discovering new antiobiotic analogs (pharmaceutical chemistry etc..)

21 Talk outline. 1. State of the art on DOM chemical composition 2. Linking DOM chemical composition and radiocarbon 3. Compound specific radiocarbon analysis (CSRA)

22 Radiocarbon composition of DOM For example a global survey of bulk DOC D 14 C values containing n>23000 samples will require > 159 years of daily UV oxidations with a cost of > $ in lab fees using today s technology

23 Radiocarbon data on DOM fractions Atlantic DOM HMWDOM > 1KDa LMWDOM < 1KDa (20-30% DOM) (70-80% DOM) 3 m 2180 y (-21.3 ) 40 y (-21.8 ) 2640 y (-21.2 ) 1500 m 3810 y (-20.8 ) 2440 y (-21.2 ) 3890 y (-20.8 ) Pacific 20 m 1800 m 1700 y (-21.2 ) 6120 y (-21.2 ) 780 y (-21.8 ) 4570 y (-21.3 ) 1890 y (-21.1 ) 6220 y (-21.2 ) a. 14 C HMWDOM younger component of DOM b. 14 C LMWDOM age is close to 14C of DOM c. All components HMWDOM, LMWDOM have a marine origin Loh et al Nature

24 Radiocarbon values in different HMWDOM fractions HMWDOM D 14 C = 46 (< 3y modern) D 14 C = -262 (2441y ) Surface Deep Hydrolysis, desalting (resins) HMWMUC Surface Deep Elution NH 4 OH Humics, recalciltrant DOM D 14 C = -416 (4321y refractory) D 14 C = -428 (4487y refractory) Rich in COOH Aliphatic component Elution H 2 O 50-70% HMWDOC Total Carbohydrates D 14 C = (< 3y modern) D 14 C = -120 (1027y) APS Surface Deep Rich in CHOH Hydrophilic component This suggests that both new (labile) and old refractory components co-exist within the HMWDOM and as such, do not support a molecular size-dependant continuum of organic carbon diagenesis (Amon and Benner, 1996). Differences in chemical composition rather than molecular size likely control differences in oceanic residence time

25 Radiocarbon analyses for old or nonreactive HMWDOC Rich in COOH Aliphatic component Repeta and Aluwihare (2006) L&O

26 Talk outline. 1. State of the art on DOM chemical composition 2. Linking DOM chemical composition and radiocarbon 3. Compound specific radiocarbon analysis (CSRA)

27 What are the comp. classes amenable for compound specific radiocarbon analyses (CSRA) in aquatic environments? Sugars: Individual monosaccharides, 2 papers (Aluwihare et al. 2002; Repeta & Aluwihare, 2006). Method HPLC-RI Amino acids: None Lipids: n-alkanes, n-alcohols, fatty acids, C:40 isoprenoids, sterols, hopanoids, alkenones etc, papers. (Method: PCGC) Nucleic acids: DNA purification, 2 papers (Cherrier et al. 1999; Hansman et al Perform CSRA in big samples (sediments, HMWDOM etc) in which we have a lot of stuff!!!!! NOT in DOM

28 Radiocarbon values in monosaccharides of the APS HMWDOM Hydrolysis desalting (resins) Elution NH 4 OH Elution H 2 O (APS) Humics and recalcitrant DOM Total Carbohydrates F4 = monosaccharides Arabinose Fucose Galactose Glucose Mannose Rhamnose Xylose F4 F3 F2 F5 F Repeta and Aluwihare (2006)

29 Laminarin DP-7 maltose DP-3 DP-5 glucose Separation of mono- and oligosaccharides in HMWDOM by ion chromatography Unhydrolyzed sugar polymer HPLC-RI conditions 2 Ag columns at 80C, loop 100 µl Eluant H 2 O, 0.5 ml/min, 40 min Neutral sugars (15-20%) Time (min)

30 Ion chromatography

31 Purification of neutral sugars from APS 1 H NMR of Fucose Fucose HMWDOM-fucose Fucose-Standard

32 Radiocarbon measurements of APS neutral sugars in the surface NPSG D 14 C ( ) DIC HMWDOC 46 Glucose 79 Galactose 103 Mannose 99 Xylose 94 Arabinose ND Fucose 69 Average sugar Repeta and Aluwihare (2006)

33 Sugar component is modern within HMWDOM Repeta and Aluwihare (2006) L&O

34 Collaboration with CEREGE for Bulk isotopic analyses (d 13 C + D 14 C) regarding POC, sediment, aerosol samples and HMWDOM Compound specific isotopic analyses (d 13 C + D 14 C) extracted from the above matrices MIO researchers involved : C.Panagiotopoulos, R. Sempéré, J-F Rontani & M. Goutx

35 Merci de votre attention