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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

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

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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


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HOW TO DETERMINE

PHYTOPLANKTON ?

peridinina

Dinoflagelados

Clorophyta

Cryptophyta

Cyanobacterias

aloxanthin



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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


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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 .


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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


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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)


Slide9 l.jpg

  • 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


    Slide10 l.jpg

  • 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 -


    Slide11 l.jpg

    chlorophyll b

    chlorophyll a

    DV-chlorophyll a

    DV-chlorophyll b

    Molecule drawings:N. Montoya


    Slide12 l.jpg

    chlorophyll c1

    chlorophyll c2

    chlorophyll c3

    Molecule drawings:N. Montoya


    Slide13 l.jpg

    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


    Slide14 l.jpg

    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


    Slide15 l.jpg

    Degradation by chemical processes:

    Loss of phytil group

    Cl chlorophyillide

    In methanol or ethanol in basic medium


    Slide16 l.jpg

    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)


    Slide17 l.jpg

    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


    Slide18 l.jpg

    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


    Slide19 l.jpg

    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


    Slide20 l.jpg

    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


    Slide21 l.jpg

    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


    Slide22 l.jpg

    DIADINOXANTHIN CICLE

    Diadinoxantin

    epoxidation

    + 2H + O2 - H2O

    DARK

    LIGHT

    + 2H - H2O

    Diatoxanthin


    Slide23 l.jpg

    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


    Slide24 l.jpg

    Xantophylls

    Isoprenoids

    Zeaxanthin

    isomers

    Lutein


    Slide25 l.jpg

    Acetil enic

    Diatoxanthin

    Alenic

    fucoxanthin

    Norcarotenoids

    ( skeleton C37)

    Peridinin

    C39H50O7


    Slide26 l.jpg

    In acid medium

    Epoxides rearrange (5,6 to 5,8 form)

    7

    6

    8

    5

    violaxanthin

    7

    6

    8

    5

    neoxanthin


    Slide27 l.jpg

    • In basic medium :

    • In general stable

    • exception:

      esters are hydrolysed

      some compounds suffer structural change (fucoxanthin, peridinin)

    fucoxanthin


    Slide28 l.jpg

    Distribution of chlorophylls among divisions/classes of phytoplankton


    Slide29 l.jpg

    Distribution of carotenes among divisions/classes of phytoplankton


    Slide30 l.jpg

    Distribution of xantophylls among divisions/classes of phytoplankton


    Slide31 l.jpg

    Distribution of xantophylls among divisions/classes of phytoplankton


    Slide32 l.jpg

    Amphidinium carterae (Dinophyta)

    Rzi =[lpigmi]/[chlorophyll a]

    Rz =[peridinin]/[chlorophyll a]

    chlorophyll c2

    chlorophyll a

    dinoxanthin

    peridinin

    diadinoxanthin


    Slide33 l.jpg

    Dunaliella tertiolecta (Chlorophyta)

    Rzi =[lpigmi]/[chlorophyll a]

    Rz =[lutein]/[chlorophyll a]

    chlorophyll b

    chlorophyll a

    neoxanthin

    violaxanthin

    lutein

    anteraxanthin



    Slide35 l.jpg

    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


    Slide36 l.jpg

    Reverse-phase HPLC eluant) of the major pigments detected in

    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


    Slide37 l.jpg

    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.



    Slide39 l.jpg

    Pigmentos sample

    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


    Slide40 l.jpg

    • DEGRADATIN PRODUCTS sample:

    • 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) .


    Slide41 l.jpg

    Chlorophyll sampleb: 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).


    Slide42 l.jpg

    Fossile Pigments sample:

    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).


    Slide43 l.jpg

    Carotenoids: sample

    Estáveis, abundantes

    (adaptado de Buchaca & Catalan 2008)


    Slide44 l.jpg

    Chlorophylls : sample

    (adaptado de Buchaca & Catalan 2008)



    Slide46 l.jpg

    Chlorophylls sample

    Phaeophytin a

    Chlorophyll a

    - Mg

    - Mg

    - Phytil

    - Mg, -COOMe

    Phaephorbide a

    Pirophaephytin a

    Jeffrey et al.;1997


    Slide47 l.jpg

    Polyene chain: chromophore sample

    UV7VIS: Electronic transitions

    Main

    transition

    Vibrational

    fine

    structure


    Slide48 l.jpg

    Calculation of % III/II for a caroteneid sample

    II

    III

    0

    0

    Vibrational

    fine

    structure


    Slide49 l.jpg

    Molecular structure x spectroscopic properties sample

    Lenght   

    Chromophore (polyene chain):


    Slide50 l.jpg

    Molecular structure x spectroscopic properties sample

    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


    Slide51 l.jpg

    Molecular environment x spectroscopic properties sample

    1: displacement of max to longer wavelength



    Slide53 l.jpg

    • HPLC method with improved resolution, LC–MS analysis and the automated acquisition of MS/MS data for pigments

    • extracts from a sediment (Priest Pot, Cumbria, UK),

    • a microbial mat (les Salines de la Trinital, South Catalonia, Spain)

    • a culture (C. phaeobacteroides):

      SEPARATION OF A GREAT NUMBER OF PIGMENTS, INCLUDING NOVEL BACTERIOCHLOROPHYLL DERIVATIVES.

    Airs, 2001


    Slide54 l.jpg

    More than 60 pigments during the run: the automated acquisition of MS/MS data for pigments

    Airs, 2001


    Slide56 l.jpg

    HPLC coupled both to UV photodiode array detection and to atmospheric pressure mass spectrometric techniques (HPLC–DAD-APIMS)

    Pigments ( chlorophylls, carotenoid), galactolipids, alkaloids, sterols and mycosporine-like amino acids,

    Frassanito 2005


    Extraction and separation of pigments l.jpg
    Extraction and separation atmospheric pressure mass spectrometric techniques (HPLC–DAD-APIMS) of pigments


    Slide59 l.jpg

    • Chemotaxonomic estimation of phytoplankton communities in aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    • Extraction must be quantitative for all pigments

    • HPLC separation must be able to:

    • separate simultaneously groups of molecules of very different polarities

    • Resolve very similar compounds, for instance isomers


    Slide60 l.jpg

    Extraction aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. of phytoplankton pigments

    Acetone 90 %

    Acetone 100 %

    Methanol

    Acetone :Methanol ( 1:1)

    N,N-dimetilformamide (DMF)

    Buffered Methanol ( 2% NH4Ac 0,5 M)

    Solvents:

    Procedure:

    Sonication or criogenic homogenization

    „overnight“ or immediate extraction


    Slide61 l.jpg

    Separation (HPLC) aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    Filtration

    GF/F 47mm

    Extration:

    Methanol: NH4Ac 0,5M (98:2) +

    Sonification, ice-bath (30 s) +

    Centrifugation (5 min, 4800 rpm)


    Slide62 l.jpg

    Chromatographic separation of aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    Phytoplankton pigments


    Slide63 l.jpg

    Separation aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. with C30 columns:Development of a

    computer-assisted method (Software Dry Lab)

    Fase estacionária:

    C30 (YMC, C30, 5µm,

    polimérica

    250x4,6 mm ID

    Fase móvel:

    A:CH3OH:TBA (28 mM)

    70:30 (v/v)

    B: CH3CH2OH

    pH 6,5

    Gradiente:

    chlorophylls:

    Fig A:30-100 % B, 50 min

    Vazão: 1,2 ml/min

    T: 47 oC

    Carotenóides:

    Fig B:25-63 % B, 35 min,

    63-100%B/13 min

    Vazão: 1,4 ml/min

    T: oC

    c3

    DV, MV cl b

    alo-, diato-xanthins

    e luteína

    c2

    DV, MV cl a

    c1

    luteína

    aloxanthin

    diatoxanthin

    Resolution:

    otimization

    for chlorophylls

    And for carotenoids in

    Sparate runs

    Mistura-teste

    Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49

    Resolution: separation mono/divynil clh a, b

    They don‘t separate in C18 !! (depends on aliphatic chain?)


    Slide64 l.jpg

    Separation with aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. C8 columns:

    1) Development of a computer-assisted method (Software Dry Lab)

    DV, MV cl b

    Zeaxanthin, luteína,

    DV, MV cl a

    c3

    Fase estacionária:

    C8 (Eclipse XDB, 3,5 µm

    150x4,6 mm ID

    Fase móvel:

    A:CH3OH:TBAA (28 mM)

    70:30 (v/v), pH 6,5

    B: CH3OH

    c1 +clorofilídeo a

    C2 +MgDVP

    Mistura-teste.Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49

    2) Zapata et al., 2000Mar. Ecol Progr. Ser. 195: 29-45, 2000

    Fase móvel:

    A:CH3OH : CH3CN : pirid.acet. (50:25:25); B: CH3OH : CH3CN : acetona (20:60:20)


    Slide65 l.jpg

    C8, Zapata aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    R=0,8

    cl b/DVcl b

    R< 0,5

    Zeaxanthin,

    dihidroluteína

    Clor c2

    R>1

    4k Hex

    /9‘cis Neo

    R> 1,25

    MgDVP

    R< 0,5

    cl b/DVcl b

    R= 0,8

    R=1

    C8, Van Heukelem

    4k Hex

    /9‘cis Neo

    Não resolve

    Pigment mixture, S. Wright, Course Notes

    C8: better for chlorophyll c family


    Slide66 l.jpg

    Comparison aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. of method sensitivity with C18 and C8 columns

    Fases estacionárias:

    C8 (Symmetry C8, 3,5 µm

    150 x 4,6 mm)

    C18 (Supelcosil L-C18, 5 µM

    250 x 4,6 mm)

    Fase móvel:

    Coluna C18:

    adap. Kraay, 1992

    A:CH3OH:H2O (85:15)

    B: CH3CN.H2O (90:10)

    C: Acet. Etila

    (vazão 0,6 ml/min)

    Coluna C8:

    Zapata, 2000

    Mendes et al., Limnol. Oceanogr. Methods 5, 2007, 363-370

    C18: More sensitivity

    Lower limit of detection

    Better for low concentration pigments


    Slide67 l.jpg

    Separation of complex samples, method aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    compatible with LC/MS

    Método SCOR 1997

    Fase estacionária:

    2 colunas „in line“

    Waters Spherisorb ODS2

    3 µM

    150 x 4,6 mm)

    Fase móvel:

    A: NH4Ac 0,01M

    B: CH3OH

    C: CH3CN

    D: Acet. Etila

    Gradiente:5%A, 85% B,

    15 % C isocr.5 min,

    0%A, 20% B,15%C,65% D,

    95 min, 0%A, 1%B, 1%C,

    98%D, 5 min,isocr. 5 min

    Adequado para

    LC/MS

    Método Airs et Al.

    Extrato de amostra de sedimento (Priest Pot)

    Airs et al.;Journal of Chromatography a 917 (2001) 167-177


    Slide68 l.jpg

    zeaxanthin aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    19,-butanoilfuco

    luteina

    Cl a + DV cla

    Cl b + DV clb

    peridinina

    diatoxanthin

    diadinoxanthin

    Cl c2

    aloxanthin

    dinoxanthin

    neoxanthin

    violaxanthin

    prasinoxanthin

    fucoxanthin

    Cl c3

    Fase estacionária:

    Spherisorb ODS1/ C18

    250 x 4,6 mm – 5 m

    Fase móvel:

    A: CH3OH 0,3 M em

    NH4Ac : ACN : H20

    (51:36:13)

    B: AcetEtila: ACN (70:30)

    Vazão: 1,2 ml/min

    Gradiente:0 a 25 % B em

    5 min, isocr.5 min,

    25% a 100% B

    em 20 min.

    Labor. UFF, Cromatógrafo Bischoffanalysentechn., Mistura-teste (DHI), 100µL injetados na fase A,

    Separates:,-carotene, ,-carotene, Aloxanthin,Lutein, Neoxanthin,

    Violaxanthin, Fucoxanthin, Diatoxanthin,Diadinoxanthin, Peridinina,

    Dinoxanthin, Zeaxanthin, Mixoxantophyll, Equinenone, Cantaxanthin,

    Astaxanthin, Okenone, Scytonemin-1, -2, Bacteriophaeophytin-a,

    Bacteriochlorophyll-e, chlorophyll-a, Chlorophilide-a, Chl-a Allomer and Epimer,

    phaeophytin- a1, a2, phaeophorbide -a1, -a2, -a3, -a3’, -a4, chlorophyll b,

    phaeophytin -b1, -b2, chlorophyll –c1, -c2, -c3

    Buchaca e Catalan (2008)


    Slide69 l.jpg

    HOW TO DETERMINE PHYTOPLANKTON ? aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    ESTIMATION OF THE ABUNDANCE OF PHYTOPLANKTONIC COMMUNITY BY PIGMENT MARKERS

    Based on the contribution, in terms of chlorophyll a,

    of each group of taxonomical class (Chl a)c

    to total chlorophyll a in the sample (Chl a)t :

    (Chl a)t = (Chl a)c1 + (Chl a)c2 + (Chl a)c3 + ...... + (Chl a)cn

    Calculation of(Chl a)cn ?

    Easy !


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    METHOD aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. 1:

    Calculation of(Chl a)c by the choice of one marker pigment

    for each class

    Problem:

    Fixed R

    not necessarily

    Corresponds

    To the ratios

    In the samples

    (Chl a)t = Rzea/cla x (Zea) + Rlut/cla x (Lut) + .........+ Rfuco x (Fuco)

    {

    sample

    (Chl a)c and

    % of each class

    Fixed


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    METHOD aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. 2:

    Multilinear regression

    Sample 1: (Chl a)t1 = Rzea/cla x (Zea)1 + Rlut/cla x (Lut)1 + .........+ Rfuco x (Fuco)1

    Sample 2: (Chl a)t2 = Rzea/cla x (Zea)2 + Rlut/cla x (Lut)2 + .........+ Rfuco x (Fuco)2

    ....................................................................................................................................

    Sample n: (Chl a)tn = Rzea/cla x (Zea)n + Rlut/cla x (Lut)n + .........+ Rfuco x (Fuco)n

    Unknown Rs, determined by pela resolution of a system

    of n equations and n unknowns

    Rs are determined, but many classes don‘t have a specific pigment

    (Chl a)cn

    % of ech class


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    M aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. ÉTODO 3:

    Determinação da composição fitoplanctônica por análise fatorial

    (MACKEY et al., 1996)

    • „Software „CHEMTAX: problema de análise fatorial:

    • matriz de dados S: concentrações encontradas para os pigmentos

    • no ambiente num conjunto de amostras

    • fatorizada em matrizes

      F : matriz das razões dos pigmentos para as diferentes classes

      de algas puras e

      C : abundâncias de cada classe de alga em cada amostra


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    MATRIZ F: aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. RazõesRi =[lpigmi]/[chlorophyll a para cada classe

    C: contribuição de cada

    classe (a ser determinada)

    MATRIZ S:experimental

    Amostra 1: (Chl a)t1 (Zea)1 (Lut)1 ....... (Fuco)1

    Amostra 2: (Chl a)t2(Zea)2 (Lut)2 ....... (Fuco)2

    .................. ............ ......... ........ .......

    Amostra n: (Chl a)tn (Zea)n (Lut) ....... (Fuco)n

    Clpras

    ClDin

    ClCryp

    ClHapt3

    ClHapt4

    ClChlor

    ClSyn

    ClDiatom

    F x C = S


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    Para uma fatoriza aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. ção de S que tenha um significado físico:

    F : variável, Fo: dados da literatura (normalizados/Cl a)

    Estimativa inicial da matriz de abundâncias das classes (Co):

    calculada resolvendo-se a equação de mínimos quadrados:

    minimizar:  S – Co Fo,

    sob as condições: [Co]ij 0  i, j

     [Co]ij = 1  j

    O resíduo é expresso por:

    o = S – Co Fo

    Um algoritmo de „decréscimo máximo“ do resíduo foi usado

    (variação dos elementos de F, 10% a cada iteração)


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    Juturnaíba reservoir as a study model aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    Marcelo Marinho e Silvana V. Rodrigues


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    OBJETIVOS aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    • Avaliar a aplicabilidade do método de análise de pigmentos por HPLC

      para detecção das variações na biomassa e composição do fitoplâncton, comparando com os dados obtidos por microscopia


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    METODOLOGIA aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    Fitoplâncton

    • Coletas quinzenais - jun/96 - mai/97 (estação central)

    • Biovolume

      • método de sedimentação (Utermöhl, 1958)

        Pigmentos

    Amostra

    (0,25 - 1,8 L)

    Injeção e análise

    HPLC

    CONDIÇÕES CROMATOGRÁFICAS

    • Filtração (GF/C)

    • Congelamento

    • (CO2 sólido)

    • Coluna C18 - fase reversa

    • Gradiente alta pressão

    • (modificado de Garrido & Zapata, 1993)

    • Detecção - 440nm

    Extração

    Metanol 100%


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    -1 aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    -1

    Biomass (chlorophyll a)

    Contribution calculated by marker pigments

    Razão Xan/Chl-a

    CHEMTAX


    Slide79 l.jpg

    • Biovolume aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    • 0,2 L +Lugol’s solution

    • sedimentation method (Utermöhl, 1958)

      • biomass: product of population and mean unit volume of each species

      • (specific density of cells = 1 g/cm3,

      • cell size = mean of at least 30 measurements)


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    Microcystis aeruginosa aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    20 mg/L

    Anabaena spiroides

    Cylindrospermopsis raciborskii

    Biomass (Biovolume)


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    Biomass (CHEMTAX) x Biomass (biovolume aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained. )

    2 periods in both methods

    CHEMTAX:

    Period 1 (June - November 96):

    3.7 - 36.4 mg/L chl a

    Chlorophyceae, Cyanobacteria, Cryptophyceae

    Period 2 (December 96- May 97):

    46.9 - 254.4 mg/L chl a

    81% to 99 % Cyanobacteria.


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    CONCLUSIONS aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    • High correlation between biovolume and Chl-a. Chl-a can be used as a parameter to estimate biovolume.

    • Interpretation of pigment data with CHEMTAX: better correlation with biovolume than that based on Xan/Chl-a ratios from unialgal cultures.

    • Only Chlorophyceae and Dinophyceae did not present significant correlation with cell count.

    • Similar general pattern of the phytoplankton community dynamics by cell count and pigment analysis: two periods and the Cyanobacteria bloom recorded.


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    GUANABARA aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    BAY

    RJ/BRAZIL

    12SAMPLING SITES:

    SAMPLING FREQUENCE:

    - 12 CAMPAIGNS

    - JANUARY TO AUGUST (SUMMER/AUTUMN) 2006


    Slide85 l.jpg

    HOMOGENEITY OF SAMPLES aquatic and sedimentary environments involves not only the choice of marker pigments, but also efficient extraction and separation procedures and a reasonable treatment of the data obtained.

    WITHIN EACH DATA MATRIX

    Data processing:

    CHEMTAX:

    Samples divided in 5

    environmentally

    different groups

    5

    2

    3

    4

    1


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