1 / 87

Determination of phytoplynkton composition and biovolume Utermöhl method: : Advantage: asy sampling, long storage times

HOW TO DETERMINE PHYTOPLANKTON?. Si lvana V. Rodrigues. Determination of phytoplynkton composition and biovolume Utermöhl method: : Advantage: asy sampling, long storage times Disadvantage: requires a lot of time, and specialists

victoria
Download Presentation

Determination of phytoplynkton composition and biovolume Utermöhl method: : Advantage: asy sampling, long storage times

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. HOW TO DETERMINE PHYTOPLANKTON? Silvana V. Rodrigues Determination of phytoplynkton composition and biovolume Utermöhl method:: • Advantage: asy sampling, long storage times • Disadvantage: requires a lot of time, and specialists • Results: relative contribution of algas classes x biovolume

  2. HOW TO DETERMINE PHYTOPLANKTON ? peridinina Dinoflagelados Clorophyta Cryptophyta Cyanobacterias aloxanthin

  3. http: //oceancolor.gsfc.nasa.gov/.../BIOLOGY/

  4. Importance of chlorophyll a • 1.000 milhão tons produzidas por ano na terra e no mar • indicator único da biomassa aquática • parâmetro bioquímico mais freqüentemente medido em oceanografia Cloroplasto fig.cox.miami.edu/.../phts/c8.10x21.overview.jpg struggle.net/history/images/ molecule.jpgwww.molecularexpressions.com

  5. Function of pigments in photosynthetic organisms chlorophyll a: light absorption (“Light harvesting complexes”) electron donor and acceptor in reative centers Carotenoids: Light absorption Protection of chlorophyll (“quenching “ of Chl photoinduced triplet state ) and quenching of O2 singlet state .

  6. Characteristics which make it possible to use algal pigments (chlorophylls, carotenoids and phycobiliproteins) as chemotaxonomic markers • They are present in all photosynthetic algae, but absent in most bacteria, protozoa and detritus • Many occur only in specific classes or even genera, allowing the determination of phytoplankton taxonomic composition at least at class level, or better • They are strongly coloured, and in the case of chlorophylls and phycobiliproteins are fluorescent, what allows their detection with high sensitivity, • Most of them are labile and esily dgraded after cell death, allowing to distinguish living from dead cells

  7. Hystorical overview • 1952: chlorophyll was recognized as a selective phytoplankton marker, in the presence of other biological components (zooplankton, bacteria, detritus) • 1984-1987: HPLC methods for the determination of chls, carotenoids and phytoplankton degradation products • Use of pigment chemotaxonomy for recognition, in field samples, of phytoplanktonic classes not detected since then, because of preservation problems or filtration losses. • alloxanthin (Cryptophyta) • chlor b (Chlorophyta and Prasinophyta) • zeaxanthin (Cyanobacteria) • 19’-hexanoiloxifucoxanthin (Prymnesiophyta) • divynil-chlorophyill a (Proclorophyta)

  8. Chlorophylls: • 132 -Metilcarboxilates of - • Mg-phytoporphyrin (double bond in D ring): Cl c, Mg-phytoclhorin: Cl a, Cl b Phytil at C-173 (Cl a and b) Propionic acid at C17: Cl a and b Acrílic acid at C17: Cl c Mg coordination complexes with cyclic tetra-pyrrols Macrocicles with five member rings

  9. Chlorophylls: • 132 -Metilcarboxilates of - • Mg-phytoporphyrin (double bond in D ring): Cl c, Mg-phytoclhorin: Cl a, Cl b Phytil at C-173 (Cl a and b) Propionic acid at C17: Cl a and b Acrílic acid at C17: Cl c Oxo substituent at C-131 methyl-carboxilate groups at C-132 -

  10. chlorophyll b chlorophyll a DV-chlorophyll a DV-chlorophyll b Molecule drawings:N. Montoya

  11. chlorophyll c1 chlorophyll c2 chlorophyll c3 Molecule drawings:N. Montoya

  12. Degradation by chemical processes: • Molecules become chemically and fotochemically • more labile in organic solvents • than in the cells • Loss of metal • Chla  Phaeophitin • in organic solvents • In dilute acids • under high intensity of light

  13. Degradation by chemical processes: • Epimerization (HPLC: in SiO2): • Allomerization (oxidation by O2): Cl  enolate  Cla’, b’ • Chl a  132 Hydroxiclhorophyll a • Chl a Cl a - Hyidroxilactone. Both processes can be minimized by decreasing the temperature In alcoholic or hydro-alcoholic solutions Specially in pH >7

  14. Degradation by chemical processes: Loss of phytil group Cl chlorophyillide In methanol or ethanol in basic medium

  15. Biodegradation: Loss of metal: Mg-dequelatase Formation of phaeophytins • To cyclic tetra-pirrols perifercally modified (enzymatically, Specially in the absence of light and O2): Decarboximetilation Formation of pirophaeophytins e pirophaeophorbides Hydrolisis of the phytil ester (chlorophyllase) chlorophillide formation Allomerization Epimerization (Chl-oxidase)

  16. Biodegradation: • To linear tetra • pirrols 5 4 Normally by oxidative opening of the macrocycle ring, between C-4 and C-5, C-5 stays as an aldehyde

  17. Carotenoids Derive from carotene: C40H56 β- β- carotene Isoprenoid units Polyen: Absorbtion of light. COLOUR -carotene: ,-carotene -carotene: ,-carotene -carotene: ,-carotene -carotene: ,-carotene lycopene: ,-carotene

  18. Properties More stable in phytoplankton and in plants than chlorophylls: they don‘t have N, so can‘t be used in enzymatic amino-acid building. Example: Leaves lose the green colour in autumn (chlorophyll), But don‘t lose colours due to carotenoids

  19. Polyene chain is responsible for instability: • Oxidation by air or peroxides • Electrophyle addition ( H+ and Lewis acids) • Isomerization E/Z caused by heat, light or chemicals, • Undergo reactions at the ends of the molecules • Production of artefacts

  20. Acetil-CoA Geranylgeranyldiphosphate Geranylgeranyldiphosphate Biosynthesis: occurs in thylakoid membranes Phytoene Dessaturation Lycopene Ciclization ,  -carotene ,  -carotene Hydroxilation Hydroxilation lutein Zeaxanthin Deepoxidation Dark Epoxidation Light Anteraxanthin Can occur in the dark Depends a lot on light Dark Epoxidation Light Deepoxidation Violaxanthin VIOLAXANTHIN CICLE Rearrangement Neoxanthin

  21. DIADINOXANTHIN CICLE Diadinoxantin epoxidation + 2H + O2 - H2O DARK LIGHT + 2H - H2O Diatoxanthin

  22. Carotenoids C40H56 β- β- carotene Aldehydes, ketones Enzimatic hydroxilation Acetates (OCOMe) e lactones Carboxi (CO2H), carbometoxi (CO2Me) ou metoxi (OMe) Hydroxi- carotenoids as fatty acid esters, or as Glycosides or glycosylesters, others as sulphates Epoxidation

  23. Xantophylls Isoprenoids Zeaxanthin isomers Lutein

  24. Acetilenic Diatoxanthin Alenic fucoxanthin Norcarotenoids ( skeleton C37) Peridinin C39H50O7

  25. In acid medium Epoxides rearrange (5,6 to 5,8 form) 7 6 8 5 violaxanthin 7 6 8 5 neoxanthin

  26. In basic medium: • In general stable • exception: esters are hydrolysed some compounds suffer structural change (fucoxanthin, peridinin) fucoxanthin

  27. Distribution of chlorophylls among divisions/classes of phytoplankton

  28. Distribution of carotenes among divisions/classes of phytoplankton

  29. Distribution of xantophylls among divisions/classes of phytoplankton

  30. Distribution of xantophylls among divisions/classes of phytoplankton

  31. Amphidinium carterae (Dinophyta) Rzi =[lpigmi]/[chlorophyll a] Rz =[peridinin]/[chlorophyll a] chlorophyll c2 chlorophyll a dinoxanthin peridinin diadinoxanthin

  32. Dunaliella tertiolecta (Chlorophyta) Rzi =[lpigmi]/[chlorophyll a] Rz =[lutein]/[chlorophyll a] chlorophyll b chlorophyll a neoxanthin violaxanthin lutein anteraxanthin

  33. Hierarchical guide to the use of pigments S. Wright, Class notes

  34. Retention times and mean absorption properties (inHPLC eluant) of the major pigments detected in Erythrobacter longus (ATCC 33941) and isolates NAP1, MG3, and NJ3Y. Peak numbers correspond to those indicated in Fig. 5. Solvents and caroteneid band ratios from the literature data: 1 solvent=methanol+ water (4:1) containing 40mM NH4OH, %(III/II)=0; 2 solvent= methanol, %(III/II)=0; 3 solvent=acetone, %(III/II)=33; 4, 5 solvent=diethyl ether; 6 solvent=acetone, %(III/II)=21 Michal Kobližek Arch Microbiol (2003) 180 : 327–338

  35. Reverse-phase HPLC chromatograms (360 nm) for acetone extracts prepared from whole cell pellets of a Erythrobacter longus ATCC 33941, b NAP1, c MG3, and d NJ3Y. Peak identities: 1 erythroxanthin sulfate, 2 bacteriorubixanthinal, 3 zeaxanthin, 4 bacteriochlorophyll a, 5 bacteriophaeophytin a, and 6 β,β-carotene Michal Kobližek Arch Microbiol (2003) 180 : 327–338

  36. HPLC chromatogram of fuorescent pigments from a surface sample (2 m depth) collected at station C354-004. Excitation was at 365 nm, emission at 780 nm, with 20-nm slits. These wavelengths were chosen to maximize the signal from BChla, while minimizing the signal from the more abundant pigments, Chla and Chlb. (Inset) Fluorescence emission spectrum of the peak eluting at 16.7 min in (A). Excitation was at 365 nm and slits were 20 nm. Zbigniew S. Kolber et al, Science 292, 2492-2495; 2001.

  37. PIGMENTS IN SEDIMENTS

  38. Pigmentos Em geral são moléculas lábeis, atingem o sedimento em vários estágios de degradação. Degradação dos pigmentos originais principalmente na água e na superfície do sedimento, durante a deposição (Hodgson et al., 1997) • Na água: • rápida e extensa • (≤95 % dos compostos em poucos dias) • digestão por herbívoros, • enzimática, na senescência celular • oxidação química, microbiológica e pela luz. Fatores que afetam a taxa de degradação: • Tempo para chegar • ao fundo • Tipo de pigmento • Grau de ataque • químico e biológico • Nos sedimentos: • taxa de degradação menor, especialmente em condições anóxicas. Depende de: • intensidade de luz e da • bioturvação invertebrada

  39. DEGRADATIN PRODUCTS: • degradation to uncoloured compounds • conversion to cis-carotenoids and phaeopigments more difficult to identify (Steenbergen et al., 1994 apud Hodgson et al., 1997). Separation and quantification of pigments in sediments More complex than in phytoplankton samples, due to the variety of degradation or transformation products (Mendes et al. 2007) .

  40. Chlorophyll b: occurs mainly ingreen algae and vascular plants, Chlorophylls c: in diatoms, dinophlagellates and some brown algae Kowalewska et al., 2004. Chl a‘ and phaeophytin: degradação products due to Environmental stress Pirophaeophitins and steril Chlorins: degradation products due to zooplankton Phaeophorbides: Degradation products due to zooplankton Jeffrey, 1997 apud Kowalewska et al., 2004).

  41. Fossile Pigments: Used in paleoclimatic and paleoenvironmental issues Chlorophylls : More labile than carotenoids , but phaephitins are persistent in sedimentary records Carotenoids: Stability depends on structure (decreases with the increase of the number of functional gruoups).

  42. Carotenoids: Estáveis, abundantes (adaptado de Buchaca & Catalan 2008)

  43. Chlorophylls : (adaptado de Buchaca & Catalan 2008)

  44. UV/VIS absorption of pigments

  45. Chlorophylls Phaeophytin a Chlorophyll a - Mg - Mg - Phytil - Mg, -COOMe Phaephorbide a Pirophaephytin a Jeffrey et al.;1997

  46. Polyene chain: chromophore UV7VIS: Electronic transitions Main transition Vibrational fine structure

  47. Calculation of % III/II for a caroteneid II III 0 0 Vibrational fine structure

  48. Molecular structure x spectroscopic properties Lenght    Chromophore (polyene chain):

  49. Molecular structure x spectroscopic properties Geometrical cis-trans isomers: small hypsochromic effect Significant hypochromic effect Reduction of vibrational fine structure Appearance of a cis-peak (≈ 142 nm below the longest maximum of the all-rans,measurd in hexane Beta-Rings: fine structure much reduced, max shorter than in the acyclic Acetylenic groups: replacement of d.bond to triple bond - 15-20 nm shorter wavelength Allenic groups Carbonyl groups Britton, 1995, Carotenoids, 3 vol, Birkhäuser

More Related