HOW TO DETERMINE

PHYTOPLANKTON?
Silvana V. Rodrigues
Determination of phytoplynkton
composition and biovolume
Utermhl method::
Advantage:

asy

sampling,

long

storage times
Disadvantage:

requires a lot of time,

and specialists
Results:

relative contribution of
algas classes x biovolume

HOW TO DETERMINE
PHYTOPLANKTON ?

peridinina

Dinoflagelados
O

CH

COO
3

OH

Clorophyta

Cryptophyta

Cyanobacterias

OH

aloxanthin

HO

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

Importance of chlorophyll a
1.000 milho tons produzidas por ano na terra e no mar
indicator nico da biomassa aqutica
parmetro bioqumico mais freqentemente medido
em oceanografia
Cloroplasto

struggle.net/history/images/
molecule.jpgwww.molecularexpressions.com

fig.cox.miami.edu/.../phts/c8.10x21.overview.jpg

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 .

diviso/classe

nome comum

gn

espc.

Algas marrons(clorofilas a e c)
Bacillariophyta

diatomceas

210

Desconh.

Dinophyta

dinoflagelados

550

4000

Crysophyta:
Chrysophyceae
Rapidophyceae

flagelados marrom-amarel.
Crysophytas,silicoflagelados
raphydophytas (cloromonadas)

120
4

1000
9

Haptophyta
Primnesiophyceae

flagelados marrom-amarel.
cocolitofordeos

50

500

Xantophyta

algas verde-amareladas

50

600

Cryptophyta

criptomonadas

>50

Eustigmatophyta

algas amarelo-esverdeadas

12

350
13
43

2500
120
650-800

10

Algas verdes (clorofilas a e b)

Chlorophyta
Clorophyceae
Prasinophyceae
Euglenophyta

Algas verdes
Flagelados verdes
Euglenoides

Algas vermelhas (clorofila a e biliprotenas)

Rhodophyta

Algas vermelhas

Algas azuis (Cyanobacteria) ( clorofila a e biliprotenas)

Cyanophyta
Prochlorophyta

Cianobactrias
proclorofitas

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

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)

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)

Acrlic acid at C17: Cl c

Propionic acid at C17: Cl a and b

Mg coordination complexes with cyclic tetra-pyrrols

Macrocicles with five member rings

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)

Acrlic acid at C17: Cl c

Propionic acid at C17: Cl a and b

Oxo substituent at C-131
methyl-carboxilate groups at C-132 -

H 2C

C H3

H 3C
N

Mg

H
H 3C

CH 3

chlorophyll a

H2C
H3C

CH3
N
N

H
H3C

Mg

H 3C

CH2

CH3
H3C H

H3C

DV-chlorophyll a

CH3
H3C H

Molecule drawings:N. Montoya

H3C

CH3

CH3

N
N
C H3

H
H

Mg

H
H3 C

O
COOCH3

H3C

CH 2

H3 C

O
COOCH3

CH 3

CH3

H3C

H 2C

H 3C

CH3
H

C H3
H

chlorophyll b

Mg

H3C

O
COOCH 3

H 3C

C H3

CH3

H3C

H2C

DVchlorophyll b

O
COOCH 3

C H3
H 3C

H 3C

H 3C

CH 3

H2 C

CH3

H3 C
N

N
Mg

H3 C

CH 3
H

O
OH

CH3

H3C
N

CH2

H2C

Mg
N

H3C

CH 3
H

Mg
N

H3 C

CH 3
H

OH
chlorophyll c2

O
COOCH 3

Molecule drawings:N. Montoya

COOCH 3
CH2

H3 C

OH

O
COOCH 3

chlorophyll c1

H2C

CH3

chlorophyll c3

O
COOCH 3

Degradation by chemical processes:

Molecules become chemically and fotochemically
more labile in organic solvents
than in the cells
H 2C

CH3

H3 C
N

Mg

H
H3 C

CH 3

Chla

N
N

Phaeophitin

in organic solvents

C H3

In dilute acids

Loss of metal

O
COOCH 3

under high intensity of light

CH3
H3C

H3C

H 3C

CH 3

Degradation by chemical processes:

H 2C

CH3

H3 C
N
N

Allomerization
(oxidation by O2):

Mg

H3 C

Epimerization
(HPLC: in SiO2):

N
C H3

H
H

CH 3

O
COOCH 3

Cl enolate Cla, b

O
Chl a 132 Hydroxiclhorophyll a
Chl a
Cl a - Hyidroxilactone.

CH3
H3C

In alcoholic or hydro-alcoholic solutions

Specially in pH >7

H3C

H 3C

CH 3

Both processes
can be minimized
by decreasing the temperature

Degradation by chemical processes:

H 2C

CH3

H3 C
N

Mg

H
H3 C

N
N
C H3

H
H

CH 3

O
COOCH 3

Loss of phytil group

Cl chlorophyillide

CH3
H3C

H3C

H 3C

CH 3

In methanol or ethanol in basic medium

Biodegradation:
To

cyclic tetra-pirrols
perifercally modified

(enzymatically,
Specially in the absence of
light and O2):

Hydrolisis of the phytil

ester
(chlorophyllase)
chlorophillide formation

H 2C

CH3

H3 C
N

Mg

H
H3 C

CH 3

Decarboximetilation

N
C H3

H
H

Loss of metal:
Mg-dequelatase
Formation of phaeophytins

Formation of
pirophaeophytins e
pirophaeophorbides

O
COOCH 3

CH3
H3C

H3C

Allomerization
Epimerization (Chl-oxidase)

H 3C

CH 3

Biodegradation:
To

linear tetra
pirrols

H 2C

CH3

H3 C

N
Normally by oxidative opening
of the macrocycle ring, between
C-4 and C-5,
C-5 stays as an aldehyde

Mg

H
H3 C

N
N
C H3

H
H

CH 3

O
COOCH 3

CH3
H3C

H3C

H 3C

CH 3

Carotenoids
Derive from carotene:

C40H56

- - carotene

Isoprenoid
units

Polyen:
Absorbtion
of light.
COLOUR
-carotene:
-carotene:
-carotene:
-carotene:
lycopene:

,-carotene
,-carotene
,-carotene
,-carotene
,-carotene

Properties
More stable in phytoplankton and in plants than chlorophylls: they dont
have N, so cant be used in enzymatic amino-acid building.

Example:
Leaves lose the green
colour in autumn
(chlorophyll),
But dont lose colours due to
carotenoids

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

Acetil-CoA

Geranylgeranyldiphosphate
Geranylgeranyldiphosphate

Biosynthesis:
occurs in
thylakoid
membranes

Phytoene
Dessaturation
Lycopene
Ciclization
, -carotene

, -carotene
Hydroxilation

lutein

Zeaxanthin
Deepoxidation
Dark
Light
Anteraxanthin
Dark
Light
Deepoxidation
Violaxanthin

Epoxidation
Epoxidation

VIOLAXANTHIN
CICLE
Rearrangement

Neoxanthin

Hydroxilation

Can occur in the dark

Depends a lot on light

DIADINOXANTHIN CICLE
Diadinoxantin

epoxidation

+ 2H + O2 - H2O
Diatoxanthin

DARK

LIGHT

+ 2H - H2O

Carotenoids

C40H56
- - carotene

Enzimatic
hydroxilation
Epoxidation

Carboxi
(CO2H),
carbometoxi
(CO2Me)
ou metoxi
(OMe)

Acetates
(OCOMe)
e lactones

Aldehydes,
ketones
Hydroxicarotenoids
as fatty acid esters,
or as
Glycosides or
glycosylesters,
others as
sulphates

Xantophylls
Isoprenoids

Zeaxanthin

isomers

Lutein

Acetilenic
Diatoxanthin

Alenic
fucoxanthin

Norcarotenoids
( skeleton C37)
Peridinin
C39H50O7

In acid medium
Epoxides rearrange (5,6 to 5,8 form)

7
6

violaxanthin

7
6

neoxanthin

In basic medium:
In general stable

exception:
esters are hydrolysed
some compounds suffer structural change (fucoxanthin, peridinin)

fucoxanthin

Distribution of chlorophylls among divisions/classes of phytoplankton

Raphidophyceae
Chrysophyceae

DVChlb

Prymnesiophyceae

DVchla

Dinophyta

MgDVP

Bacillariophyta

Tipo pyhtilat.
Chlc

Eustimatophyta

Chl c3

Euglenophyta

Chl c2

Prasinophyceae

Chl c1

Chlorophyceae

Chlb

Cryptophyta

Chl a

Rhodophyta

Pigment

Prochlorophyta

Cyanophyta

Division or
class

Distribution of carotenes among divisions/classes of phytoplankton

Raphidophyceae
Chrysophyceae
Prymnesiophyceae
Dinophyta
Bacillariophyta
Eustimatophyta

Euglenophyta

Prasinophyceae

Chlorophyceae

Cryptophyta

Rhodophyta

Pigment

Prochlorophyta

Cyanophyta

Division or
class

Distribution of xantophylls among divisions/classes of phytoplankton

Astaxanthin
19-Butanoilfucoxanthin
Cantaxanthin

Crocoxanthin
Diadinoxanthin
Diatoxanthin
Dinoxanthin
Echinenona
Fucoxanthin

2
1

Raphidophyceae

Anteraxanthin

Chrysophyceae

Aloxanthin

Prymnesiophyceae

Dinophyta

Euglenophyta

Bacillariophyta

Prasinophyceae

Eustimatophyta

Chlorophyceae

Cryptophyta

Rhodophyta

Pigment

Prochlorophyta

Cyanophyta

Division or class

Distribution of xantophylls among divisions/classes of phytoplankton

Peridininol
Prasinoxanthin
Pirroxanthin

Violaxanthin
Zeaxanthin

14

14

Raphidophyceae

Peridinina

Chrysophyceae

P457+P468

Prymnesiop.

Neoxanthin

st. Vaucheriax

Dinophyta

Monadoxanthin

Sifoneina

Bacillariophyta

Lutena

Sifonaxanthin

Eustimatophyta

19hexanoilfuco

Euglenophyta

Prasinophyceae

Chlorophyceae

Cryptophyta

Rhodophyta

Pigment

Prochlorophyta

Cyanophyta

Division or
class

Amphidinium carterae (Dinophyta)

Rzi =[lpigmi]/[chlorophyll a]

Rz =[peridinin]/[chlorophyll a]

chlorophyll c2

chlorophyll a
dinoxanthin

peridinin

diadinoxanthin

Dunaliella tertiolecta (Chlorophyta)

Rz =[lutein]/[chlorophyll a]

Rzi =[lpigmi]/[chlorophyll a]

chlorophyll b

chlorophyll a

neoxanthin
violaxanthin

anteraxanthin

lutein

Hierarchical guide to the use of pigments

Pigment
Chl a:

Significance
an index of total algal biomass, excluding
prochlorophytes.

Unambiguous markers for algal types

DV-Chl a:
DV-Chl b:
Siphonaxanthin esters:

an index of prochlorophyte biomass

unambiguous marker for prochlorophytes
unambiguous marker for Type 2 prasinophytes
(Egeland et al., 1997)
Prasinoxanthin:
unambiguous marker for Type 3 prasinophytes
Peridinin:
Type 1 dinoflagellates
Alloxanthin:
Cryptophytes
Gyroxanthin diester:
Dinoflagellates Type 2
Chl c2 MGDG [14:0/14:0]: Chrysochromulina spp. (Haptophyte Type 7, Zapata
et al., 2004)

S. Wright, Class notes

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
Pea
k
no.

Rt

Pigmentidentification Observed
max

(min)

(nm)

Published
max

Reference

(nm)

11.4

Erythroxanthinsulfate 465

469

Takaichietal.(1991)

18.4

Bacteriorubixanthinal

513

510

Takaichietal.(1988)

19.1

Zeaxanthin

(428),454,482 (428),454,481

20.4

Bacteriochlorophylla

359,580,771

358,577,773

Scheer(1991)

23.4

Bacteriophaeophytin
a

358,525,750

357,525,749

Scheer(1991)

25.4

,carotene

(426),454,478 (426),454,480

Michal Kobliek Arch Microbiol (2003) 180 : 327338

Jeffreyetal.(1997)

Jeffreyetal.(1997)

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 ,-carotene

Michal Kobliek Arch Microbiol (2003) 180 : 327338

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.

PIGMENTS IN SEDIMENTS

Pigmentos
Em geral so molculas lbeis, atingem o sedimento em vrios estgios de degradao.

Degradao dos pigmentos originais

principalmente na gua e na superfcie do sedimento, durante a deposio
(Hodgson et al., 1997)

Na gua:
rpida e extensa
(95 % dos compostos em poucos dias)
digesto por herbvoros,
enzimtica, na senescncia celular
oxidao qumica, microbiolgica e
pela luz.
Nos sedimentos:
taxa de degradao menor, especialmente
em condies anxicas. Depende de:
intensidade de luz e da
bioturvao invertebrada

Fatores que afetam

a taxa
de degradao:

Tempo para chegar

ao fundo
Tipo de pigmento
Grau de ataque
qumico e biolgico

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) .

Chlorophyll b: occurs mainly ingreen algae and vascular plants,

Chlorophylls c: in diatoms, dinophlagellates and some brown algae

Kowalewska et al., 2004.

Phaeophorbides:
Degradation products due
to zooplankton

Chl a and phaeophytin:

degradao products due to
Environmental stress

Pirophaeophitins and steril

Chlorins: degradation
products due to zooplankton

Jeffrey, 1997 apud Kowalewska et al., 2004).

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).

Carotenoids:
Pigmento

Grupos
Funcionais

Afinidade taxonmica

b,b-caroteno

Cianobactrias, algas eucariticas e plantas vasculares

b,e-caroteno

Criptofitas

Aloxantina

Cryptofitas

Luteina

Clorfitas

Neoxantina

Clorfitas

Violaxantina

Chrisofitas e Clorfitas

Fucoxantina

Chrisofitas e Diatomceas

Diatoxantina

Diatomceas

Diadinoxantina

Dinoflagelados, Crisofitas e Diatomceas

Peridinina

Dinoflagelados

Dinoxantina

Dinoflagelados

Zeaxantina

Cianobactrias, Clorfitas

Myxoxantofila

Cianobactrias

Echinenona

Cianobactrias e zooplncton (Cladocera)

Cantaxantina

Cianobactrias e zooplncton (Cladocera)

Astaxantina

Zooplncton (Crustacea)

Okenona

Bactrias fotossintticas (Chromatiaceae)

Scytonemina-1, -2

Organismos fotossintticos expostos a alta radiao UV

Estveis, abundantes

(adaptado de Buchaca & Catalan 2008)

Chlorophylls :
Pigmento

Afinidades taxonmicas

Bacteriofeofitina-a

Bactrias fotossintticas (Rodospirillaceae e Chromatiaceae)

Bacterioclorofila-e

Bactrias fotossintticas (variedades marrons de Chlorobiaceae)

Clorofila-a

Razo molar Cl-a/forbinas a como indicador de preservao

Chlorofildeo-a

Produto de degradao da Cl-a, abundante em Diatomceas

Cl-a (almero)

Produto de degradao da Cl-a

Cl-a (epmero)

Produto de degradao da Cl-a

Feofitina-a1, -a2

Produto de degradao da Cl-a (senescncia)

Feoforbdeo-a1, -a2,

Produto de degradao da Cl-a (grazing)

-a3, -a30, -a4

Clorofila-b

Clorfitas

Feofitina-b1, -b2

Produto de degradao da Cl-b

Clorofila-c1

Crisofitas e Diatomceas

Clorofila-c2

Crisofitas, Diatomceas, Criptofitas e Dinoflagelados

Clorofila-c3

Crisofitas e Diatomceas
(adaptado de Buchaca & Catalan 2008)

UV/VIS absorption of pigments

Chlorophylls
Phaeophytin a
Chlorophyll a

- Mg

- Mg - Phytil

- Mg, -COOMe

Phaephorbide a
Pirophaephytin a

Jeffrey et al.;1997

Polyene chain: chromophore

UV7VIS: Electronic transitions

Main
transition

Vibrational
fine
structure

Calculation of % III/II for a caroteneid

II
0

III
Vibrational
fine
structure

Molecular structure x spectroscopic properties

Chromophore (polyene chain):
carotenoid

Conjug.
db. bonds

Lenght
max

phytoene

(hexane)
276 286 297

-carotene

378 400 425

lycopene

11

444 470 502

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, Birkhuser

Molecular environment x spectroscopic properties

Solvent

Approx. bathochromic shift1

Hexane, light petroleum,

ethanol, diethylether,
acetonitrile

acetone

2-6

chloroform

10-20

dichlorometane

10-20

benzene

18-24

toluene

18-24

pyridine

18-24

Carbon disulphide

18-24

1: displacement of max to longer wavelength

Identification of pigments by Mass Spectrometry

HPLC method with improved resolution, LCMS 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

More than 60 pigments during the run:

QuickTime and a
decompressor
are needed to see this picture.

Airs, 2001

QuickTime and a
decompressor
are needed to see this picture.

HPLC coupled both to UV photodiode array detection

and to atmospheric pressure mass spectrometric
techniques (HPLCDAD-APIMS)

QuickTime and a
decompressor
are needed to see this picture.

Pigments ( chlorophylls, carotenoid), galactolipids, alkaloids, sterols and

mycosporine-like amino acids,

Frassanito 2005

QuickTime and a
decompressor
are needed to see this picture.

Extraction and separation

of pigments

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

Extraction of phytoplankton pigments

Solvents:

Acetone 90 %
Acetone 100 %
Methanol
Acetone :Methanol ( 1:1)
N,N-dimetilformamide (DMF)
Buffered Methanol ( 2% NH4Ac 0,5 M)

Procedure:

Sonication or criogenic homogenization

overnight or immediate extraction

Filtratio
n

Separation
(HPLC)

GF/F 47mm

Extration:
Methanol: NH4Ac
0,5M (98:2) +
Sonification,
ice-bath (30 s) +
Centrifugation (5
min, 4800 rpm)

Chromatographic separation of
Phytoplankton pigments

Fase estacionria:
C30 (YMC, C30, 5m,
polimrica
250x4,6 mm ID

Separation with C30 columns: Development of a

computer-assisted method (Software Dry Lab)

c3

Fase mvel:
A:CH3OH:TBA (28 mM)
70:30 (v/v)
B: CH3CH2OH
pH 6,5

c1c2

alo-, diato-xanthins
e lutena

DV, MV cl b

DV, MV cl a

Resolution:
otimization
for chlorophylls
And for carotenoids in
Sparate runs

lutena

chlorophylls:
Fig A:30-100 % B, 50 min
Vazo: 1,2 ml/min
T: 47 oC
Carotenides:
Fig B:25-63 % B, 35 min,
63-100%B/13 min
Vazo: 1,4 ml/min
T: oC

aloxanthin
diatoxanthin

Gradiente:

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

Resolution: separation mono/divynil clh a, b

They dont separate in C18 !! (depends on aliphatic chain?)

Separation with C8 columns:

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

C8 (Eclipse XDB, 3,5 m

150x4,6 mm ID

Fase mvel:

C2 +MgDVP

Fase estacionria:

c3

DV, MV cl b

c1 +clorofildeo a

Zeaxanthin, lutena,

DV, MV cl a

A:CH3OH:TBAA (28 mM)

70:30 (v/v), pH 6,5
B: CH3OH

Mistura-teste.Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 3149

2) Zapata et al., 2000

Mar. Ecol Progr. Ser. 195: 29-45, 2000

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

C8, Zapata

R=0,8
Zeaxanthin,
dihidrolutena

Clor c2

R>1

4k Hex
/9cis Neo
R> 1,25

MgDVP

R< 0,5

C8, Van Heukelem

cl b/DV cl b
R< 0,5

R=1

cl b/DV cl b
R= 0,8

4k Hex
/9cis Neo
No resolve

Pigment mixture, S. Wright, Course Notes

C8: better for chlorophyll c family

Comparison of method sensitivity with C18 and C8 columns

Fases estacionrias:
C8 (Symmetry C8, 3,5 m
150 x 4,6 mm)
C18 (Supelcosil L-C18, 5 M
250 x 4,6 mm)

Fase mvel:
Coluna C18:
adap. Kraay, 1992
A:CH3OH:H2O (85:15)
B: CH3CN.H2O (90:10)
C: Acet. Etila
(vazo 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

Separation of complex samples, method

compatible with LC/MS
Mtodo SCOR 1997
Fase estacionria:
2 colunas in line
Waters Spherisorb ODS2
3 M
150 x 4,6 mm)

Fase mvel:
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

Mtodo Airs et Al.

Adequado para
LC/MS
Extrato de amostra de sedimento (Priest Pot)
Airs et al.; Journal of Chromatography a 917 (2001) 167-177

Cl a + DV cla

Cl b + DV clb

diadinoxanthin
dinoxanthin
aloxanthin
diatoxanthin
luteina
zeaxanthin

peridinina
19,-butanoilfuco
fucoxanthin
neoxanthin
prasinoxanthin
violaxanthin

Cl c2
Cl c3

Fase estacionria:
Spherisorb ODS1/ C18
250 x 4,6 mm 5 m

Fase mvel:
A: CH3OH 0,3 M em
NH4Ac : ACN : H20
(51:36:13)
B: AcetEtila: ACN (70:30)
Vazo: 1,2 ml/min
Gradiente:0 a 25 % B em
5 min, isocr.5 min,
25% a 100% B
em 20 min.

Labor. UFF, Cromatgrafo Bischoffanalysentechn., Mistura-teste

(DHI), 100L 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)

HOW TO DETERMINE PHYTOPLANKTON ?

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 =

Easy !

(Chl a)c1 + (Chl a)c2 +

...... + (Chl a)cn

Calculation of (Chl a)cn ?

(Chl a)c3 +

METHOD 1:
Calculation of (Chl a)c by the choice of one marker pigment
for each class
Class

Marker pigment (Pm)

Pm/Cla ratio in the class

Cianobactrias

zeaxanthin

Rzea/cla

Clorophyta

lutena

Rlut/cla

Dinophyta

peridinina

Rper/cla

Cryptophyta

aloxanthin

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

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

Bacyllariophyta

fucoxanthin

Fixed

Rfuco/cla

Rzea/cla x (Zea) + Rlut/cla x (Lut) +

+ Rfuco x (Fuco)

(Chl a)t =

Problem:
Fixed R
not necessarily
Ralo/cla
Corresponds
....................... To the ratios
In the samples

(Chl a)c and sample

% of each class

.........

METHOD 2:
Multilinear regression
Sample 1:
+

(Chl a)t1 =

Rzea/cla x (Zea)1

Rlut/cla x (Lut)1

Rzea/cla x (Zea)2

Rlut/cla x (Lut)2

.........+ Rfuco x (Fuco)1

Sample 2:

(Chl a)t2 =

+ .........+ 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

(Chl a)cn
% of ech class

Rs are determined, but

many classes dont
have a specific
pigment

MTODO 3:
Determinao da composio fitoplanctnica por anlise fatorial
(MACKEY et al., 1996)

Software CHEMTAX: problema de anlise fatorial:

matriz de dados S: concentraes encontradas para os pigmentos
no ambiente num conjunto de amostras
fatorizada em matrizes
F : matriz das razes dos pigmentos para as diferentes classes
de algas puras e
C : abundncias de cada classe de alga em cada amostra

MATRIZ F: Razes Ri =[lpigmi]/[chlorophyll a para cada classe

PER

BUT

FUC

HEX

NEO

PRA

VI0L

ALO

LUT

ZEA

CLB

CLA

Prasinophyt
a

0,061

0,127

0,004

0,381

0,403

Dinophyta

0,515

0,485

Cryptophyta

0,186

0,814

Haptophyta3

0,630

0,370

Haptophyta4

0,104

0,247

0,227

0,422

Chorophyta

0,040

0,035

0,127

0,006

0,165

0,628

Synecho.

0,258

0,742

Diatomaceas

0,430

00

0,570

C: contribuio de cada

MATRIZ S: experimental
Amostra 1:
Amostra 2:

(Chl a)t1
(Chl a)t2

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

Amostra n:

(Chl a)tn

classe (a ser determinada)

(Zea)1
(Lut)1
.......
(Zea)2
(Lut)2
.......
............
.........
(Zea)n
(Lut)
.......

FxC=S

(Fuco)1
Clpras
(Fuco)2
ClDin
........
ClCryp
(Fuco)n
ClHapt3
ClHapt4
ClChlor
ClSyn
Cl

....
...

Para uma fatorizao de S que tenha um significado fsico:

F : varivel, Fo: dados da literatura (normalizados/Cl a)
Estimativa inicial da matriz de abundncias das classes (Co):
calculada resolvendo-se a equao de mnimos quadrados:
minimizar:

S Co Fo

sob as condies: [Co]ij 0 i, j

[Co]ij = 1 j
O resduo expresso por:

o =

S Co Fo

Um algoritmo de decrscimo mximo do resduo foi usado

(variao dos elementos de F, 10% a cada iterao)

Juturnaba reservoir as a
study model
42

23

Rio de Janeiro
State

QuickTime and a
decompressor
are needed to see this picture.

MarceloMarinhoeSilvanaV.Rodrigues

OBJETIVOS

Avaliar

a aplicabilidade do mtodo de anlise de pigmentos por HPLC

para deteco das variaes na biomassa e composio do fitoplncton,

comparando com os dados obtidos por microscopia

METODOLOGIA
Fitoplncton
Coletas quinzenais - jun/96 - mai/97 (estao central)
Biovolume
mtodo de sedimentao (Utermhl, 1958)

Pigmentos
Amostra
(0,25 - 1,8 L)

Filtrao (GF/C)
Congelamento
(CO2 slido)

Injeo e anlise
HPLC
CONDIES
CROMATOGRFICAS
Coluna C18 - fase reversa
Gradiente alta presso
(modificado de Garrido & Zapata, 1993)

Extrao
Metanol 100%

Deteco - 440nm

Biomass(chlorophylla)
Contribution calculated by marker pigments
CHEMTAX

-1

-1

Razo Xan/Chl-a

Biovolume
0,2 L +Lugols solution
sedimentation method (Utermhl, 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)

Biomass(Biovolume)
90

mg/L
cyanobacteria
diatoms
cryptomonads

60

dinoflagellates
green algae
30

others

0
Jun

Jul

Aug

Sep
Oct
1996

Nov

Nov

Dec

Jan

Feb

Mar
1997

Apr

Microcystis aeruginosa

May

20 mg/L

Anabaena spiroides
Cylindrospermopsis raciborskii

Percentages of phytoplankton assemblages as dominant groups

of species, by period in Juturnaba Reservoir.
Period 1

Period 2a

Period 2b

12 Jun - 10 Dec

26 Dec - 17 Apr

30 Apr - 28 May

24% A. distans

72% M. aeruginosa

46% C. raciborskii

21% Cryptomonas sp.

11% A. spiroides

42% A. spiroides

Correlations between contributions of the classes found by

pigment data and by biovolume calculation (significant *p
< 0.05, **p < 0.01; n = 25).
Ratio Xan/Chl-a
Dinophyceae
Bacillariophyceae
Cryptophyceae
Chlorophyceae
Cyanobacteria
Biovolume total

0.20
0.64*
0.39
0.39
0.89**
0.97**

CHEMTAX
0.27
0.76**
0.73**
-0.35
0.97**
0.97**

Biomass (CHEMTAX) x Biomass (biovolume)

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.

CONCLUSIONS
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.

12 SAMPLING SITES:
SAMPLING FREQUENCE:
- 12 CAMPAIGNS
- JANUARY TO AUGUST
(SUMMER/AUTUMN) 2006

GUANABARA
BAY
RJ/BRAZIL

HOMOGENEITY OF SAMPLES
WITHIN EACH DATA MATRIX

Data processing:
CHEMTAX:
Samples divided in 5
environmentally
different groups

5
3
4