Production dynamics of cyanopeptides by Microcystis aeruginosa and Dolichospermum flos-aquae and effects of nutrient availability Regiane Natumi1,, Elisabeth M.L. Janssen1* 1 Department of Environmental Chemistry, Swiss Federal Institute of Aquatic Science and Technology (Eawag), 8600 Dübendorf, Switzerland Electronic Supplementary Information The electronic supplementary information contains 49 pages numbered S1-S49, 13 figures, and 7 tables. S 1 List of Tables Table S1. Composition of modified WC growth medium (Guillard and Lorenzen, 1972). ................................................................................................................... 5 Table S2. Standard analytical information including: absolute response factor (RF), relative response factor (RRF), matrix effect (ME) for Microcystis aeruginosa PCC7806 (PCC7806) and for Dolichospermum flos-aquae (Doli), limit of detection (LOD), limit of quantification (LOQ) and recovery from the SPE clean-up and concentration steps for all cyanopeptide for which a reference standard or bioreagent was available. .................................................................. 7 Table S3. Relative response factor (RRF) to microcystin-LR and extraction method recovery in percent for the 10 most abundant cyanopeptides identified in extracts of Microcystis aeruginosa or Dolichospermum flos-aquae for which no reference standard or bioreagent was available. .................................................. 8 Table S4. Cyanopeptide suspect list with compound name, compound class, neutral molecular formula, and monoisotopic mass of M+H. The table is placed at the end of the document. ............................................................................................ 8 Table S5. List of all tentatively identified cyanopeptides in cell extracts of Microcystis aeruginosa PCC7806. Only the peptides that could be classified as tentative candidate (level 3), probable structure (level 2) or confirmed structure (level 1) are reported. ......................................................................................... 18 Table S6. List of all tentatively identified cyanopeptides in cell extracts of Dolichospermum flos-aquae. Only the peptides that could be classified as tentative candidate (level 3), probable structure (level 2) or confirmed structure (level 1) are reported. ......................................................................................... 19 Table S7. Isobaric compound group name and individual compounds within each group with the same molecular formula. ........................................................... 20 S 2 List of Figures Figure S1. Correlation between optical density at 750nm and cell density (cells L-1) for three N:P conditions (N:P =2, N:P= 20, N:P= 40). ........................................ 6 Figure S2. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of microcystin- LR (top) and Microcystis aeruginosa biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted. ................................................................................................................... 9 Figure S3. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of cyanopeptolin A (top) and Microcystis aeruginosa biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted. ................................................................................. 10 Figure S4. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of cyanopeptolin D (top) and Microcystis aeruginosa biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted. ................................................................................. 11 Figure S5. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of aerucyclamide A (top) and Microcystis aeruginosa biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted. ................................................................................................................. 12 Figure S6. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of oscillamide Y (top) and Dolichospermum flos-aquae biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted. ................................................................................................................. 13 Figure S7. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of Anabaenopeptin NZ857 (top) and Dolichospermum flos-aquae biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted. Both, Anabaenopeptin NZ857 and Oscillamide Y (see Figure S6), are isobaric compounds with the same m/z and RT. Here, the fragmentation spectrum of the biomass extract does not match with the fragmentation spectra of the bioreagent Anabaenopeptin NZ857 and we conclude that Anabaenopeptin NZ857 was not present at our samples. ...... 14 Figure S8. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of anabaenopeptin A (top) and Dolichospermum flos-aquae biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted. ....................................................................... 15 Figure S9. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of anabaenopeptin B (top) and Dolichospermum flos-aquae biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the HCD collision energy are noted.a Several CID and HCD collision energies were S 3 tested (CID 30 and 35; HCD 10, 20, 30, 40 and 50), however a better mass spectrometry fragmentation spectra could not be obtained for this compound. 16 Figure S10. Comparison of intensity over m/z range for mass spectrometry fragmentation spectra between confirmed structures (Level 1) of microcystin- YR (top) and Dolichospermum flos-aquae biomass extract (bottom) in head to tail plots. The m/z value, the retention time (min) and the CID collision energy are noted. ............................................................................................................ 17 Figure S11. Non-normalized growth curve of Microcystis aeruginosa PCC7806 under different N:P ratios in the WC-Medium. ................................................. 21 Figure S12. Total cyanopeptide concentrations in µmol L-1 as cyanopeptide class equivalents relative to total cell abundance for the cyclamides (left), microcystins (middle), and cyanopeptolins (right). The slope represents the cyanopeptide production rate in fmol cell-1 and parameters of the linear regression are listed in the insert........................................................................ 22 Figure S13. Total cyanopeptide concentrations in µmol L-1 cyanopeptide class equivalent relative to total cell abundance over the growth curve for three nutrient conditions N:P 2, 20 and 40. ................................................................ 23 S 4 Table S1. Composition of modified WC growth medium (Guillard and Lorenzen, 1972). Components Control +P(x10) -P(:2) +P -P -N -N and –P -MN (mg L-1) (x3) (:100) (:100) (:100) (:100) N/P=20 N/P=2 N/P=40 K2HPO4.3H2O 11.4 114 5.7 34.2 0.114 11.4 0.114 11.4 NaNO3 85 85 85 85 85 0.85 0.85 85 CaCl2.2H2O 36.8 36.8 36.8 36.8 36.8 36.8 36.8 36.8 MgSO4.7H2O 37 37 37 37 37 37 37 0.37 NaHCO3 12.6 12.6 12.6 12.6 12.6 12.6 12.6 12.6 Na2EDTA 4.36 4.36 4.36 4.36 4.36 4.36 4.36 0.0436 FeCl3.6H2O 3.15 3.15 3.15 3.15 3.15 3.15 3.15 0.0315 CuSO4.5H2O 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.0001 ZnSO4.7H2O 0.022 0.022 0.022 0.022 0.022 0.022 0.022 0.00022 CoCl2.6H2O 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.0001 MnCl2.4H2O 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.0018 Na2MoO4.2H2O 0.006 0.006 0.006 0.006 0.006 0.006 0.006 0.00006 H3BO3 1.00 1.00 1.00 1.00 1.00 1.00 1.00 0.01 TES buffer 115 115 115 115 115 115 115 115 S 5 2.0E+09 8.0E+08 2.0E+09 N:P 2 N:P 20 N:P 40 1.6E+09 6.0E+08 1.5E+09 1 1 - 1.2E+09 1 - - L L L s s l 4.0E+08 s l l l 1.0E+09 l l e e e c c 8.0E+08 c 2.0E+08 y = 2E+09x + 5E+07 5.0E+08 4.0E+08 y = 2E+09x + 2E+08 R² = 0.7676 y = 2E+09x + 2E+08 R² = 0.9012 R² = 0.7885 0.0E+00 0.0E+00 0.0E+00 0 0.25 0.5 0.75 1 0 0.1 0.2 0.3 0.4 0 0.2 0.4 0.6 0.8 O.D. (750nm) O.D. (750nm) O.D. (750nm) Figure S1. Correlation between optical density at 750nm and cell density (cells L-1) for three N:P conditions (N:P =2, N:P= 20, N:P= 40). S 6 Table S2. Standard analytical information including: absolute response factor (RF), relative response factor (RRF), matrix effect (ME) for Microcystis aeruginosa PCC7806 (PCC7806) and for Dolichospermum flos-aquae (Doli), limit of detection (LOD), limit of quantification (LOQ) and recovery from the SPE clean-up and concentration steps for all cyanopeptide for which a reference standard or bioreagent was available. Cyanopeptide RF RRF (MCLR) ME PCC7806 (%) ME Doli (%) LOD (µg/L) LOQ µg/L) Recovery (%) Microcystin LR 2.34E+10 1 9.43 6.55 1.58 5.26 85.84 Microcystin LY 1.54E+10 0.66 4.78 4.35 1.43 4.76 65.61 Microcystin LF 1.18E+10 0.51 3.63 1.52 1.24 4.12 61.54 Microcystin LA 2.84E+10 1.22 3.83 2.28 1.35 4.50 69.45 Microcystin LW 1.81E+10 0.78 5.05 7.64 1.07 3.55 16.74 Microcystin YR 1.89E+10 0.81 6.50 9.38 1.70 5.65 68.69 Microcystin RR 5.44E+10 2.33 1.95 0.85 1.52 5.06 63.49 [D-Asp3,E-Dhb7]MC-RR 5.36E+10 2.29 2.87 1.39 1.65 5.49 n.a.