Characterization of sinking particles from twilight zone using advanced solid-state NMR Zhanfei Liu 1, Jingdong Mao 1, Michael L. Peterson 2, Cindy Lee 3, Stuart G. Wakeham 4, Patrick G. Hatcher 1 1 Old Dominion University, Department of Chemistry and Biochemistry, Norfolk, VA School of Oceanography, University of Washington, Seattle, WA Marine Sciences Research Center, Stony Brook University, Stony Brook, NY Skidaway Institute of Oceanography, 10 Ocean Science Circle, Savannah, GA Methods and sampling stations MedFlux is supported by the NSF Chemical Oceanography Program, and we also thank the Frank Batten Endowment to ODU. Introduction and motivation Sinking particles are the major vehicle for transporting carbon from surface to deep ocean. During this transit, their chemical composition changed dramatically in the “twilight zone” at depths of m, with a major fraction of the particles becoming “uncharacterizable” by conventional chromatographic analysis (Lee et al., 2004). However, the exact mechanism controlling this dramatic chemical alteration is unclear. Moreover, we still do not fully understand the factors controlling the sinking velocity of the sinking particles, i.e., which factor is more important, mineral contents or organic components? To address these questions, here we applied advanced NMR techniques including CP/TOSS, 2D HETCOR and T1 inversion recovery on the sinking particles collected from the twilight zone in the Mediterranean Sea. Fig. 4. CP/TOSS 13C NMR spectra of sinking particles from different depths in the twilight zone. The 200m sample was from the stage 1 particles by the NetTrap-elutriator, while the 520 and 920m particles were collected by IRS sediment traps. Fig. 3. Two dimensional 1 H- 13 C HETCOR of sinking particle Stage 1. Conclusions and future work  Sampling sites: DYFAMED site in the Mediterranean Sea;  Sampling tools: particles from 200m were collected by NetTraps, and those from 520 and 920m by indented rotating sphere (IRS) sediment traps. The 200m particles were further separated into four fractions with different sinking velocities, using an elutriator (Peterson et al., 2005);  Analytical techniques: sinking particles were directly analyzed by a solid-state 400 MHZ NMR spectrometer (Bruker). The advanced techniques applied on the particles included 13 C cross polarization and total suppression of sidebands (CP/TOSS), two-dimensional 1 H- 13 C heteronuclear correlation (HETCOR), and T1 inversion recovery. Fig. 1. Sampling site and the elutriator used to process the sinking particles, which were separated into 4 stages with different sinking velocities. Results and discussions 1. Comparison of sinking particles with different sinking velocities The sinking particles from 200m were dominated by lipids, carbohydrates and proteins, and the four stages separated by the elutriator showed similar chemical compositions, consistent with our conventional chromatographic analysis (Goutx et al., 2007). Fig. 2. CP/TOSS 13 C NMR spectra of sinking particles from 4 stages with different sinking velocities. The 2D HETCOR results of Stage 1 particles (200m) showed that lipids, polysaccharides and proteins were well separated from each other, and exist as discrete biopolymer domains. This is consistent with the fact they are mainly from fresh planktonic matter. 2. Comparison of sinking particles from different depths (CP/TOSS) The spectrum of the sinking particles from 200m showed typical biopolymer characteristics with sharp peaks, while in deeper depths of 520 and 920m, the peaks were greatly broadened. Moreover, the carbohydrate (67-113ppm) portion in deeper depths was significantly enhanced relative to other components. These results suggest that the fresh biopolymers are becoming more homogenous with depth due to the decomposition, and the polysaccharides are more resistant than proteins and lipids. 3. Comparison of sinking particles from different depths (T1 inversion) Fig C NMR T1 inversion recovery with different delay time (ms). a) 200m sinking particles; b) 920m sinking particles. References Goutx et al., Limnol. Oceanogr. 52(4), , 2007 Lee et al., Ambio 33, , 2004 Peterson et al., Limnol. Oceanogr.: Methods 3, , )The sinking particles from 200m had much longer recovery time than those from 920m. 2)For the 200m particles, proteins recovered much faster than carbohydrates and lipids; For the 920m particles, all the chemical components recovered at about the same rates. 3)These results suggest that organic molecules in sinking particles become more homogenous and are tangled more closely with the depth. 1. Particles with different sinking velocities have similar chemical compositions. 2. Sinking particles from surface ocean showed typical biopolymer properties, existing as separate domains of proteins, carbohydrates and lipids. In the deeper depths, polymer domains seem to disappear and become more homogeneous due to degradation. 3. Test of mineral effect on the NMR results is under way. Stage 1 (>230m/d) Stage 2 ( m/d) Stage m/d Stage m/d 200m 520m 920m “proteins” “carbohydrates” “lipids” Stage 1 Stage 2 Stage 3 Stage 4 proteinscarbohydrateslipids 1ms 110ms 85ms 75ms 70ms 65ms a. 200m 70ms 30ms 10ms 1ms b. 920m