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
HOW TO DETERMINE peridinina O

PHYTOPLANKTON ? O
O
CH 3COO OH Dinoflagelados

Clorophyta

Cryptophyta
Cyanobacterias
OH

aloxanthin

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

struggle.net/history/images/ fig.cox.miami.edu/.../phts/c8.10x21.overview.jpg
molecule.jpgwww.molecularexpressions.com
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 .
divisão/classe nome comum gên esp
Algas marrons(clorofilas a e c)
Bacillariophyta diatomáceas 210 Des
Dinophyta dinoflagelados 550 400
Crysophyta: flagelados marrom-amarel.
Chrysophyceae Crysophytas,silicoflagelados 120 100
Rapidophyceae raphydophytas (cloromonadas) 4 9
Haptophyta flagelados marrom-amarel.
Primnesiophyceae cocolitoforídeos 50 500
Xantophyta algas verde-amareladas 50 600
Cryptophyta criptomonadas 8 >50
Eustigmatophyta algas amarelo-esverdeadas 6 12
Algas verdes (clorofilas a e b)
Chlorophyta
 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)

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
 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 -
 H2C H2C H O
CH3
CH3 CH3
H3C H3C
N N N N
Mg Mg
chlorophyll a N chlorophyll b
H N N H N
CH3 CH3
H3C H3C
H H
H O
H O O
O COOCH 3
COOCH 3
O CH3
O CH3
H3C H3C H H3C
H
CH3
H3C H3C H H3C
H
CH3

H2C H2C H O
CH3
CH2 CH2
H3C H3C
N N N N
Mg Mg
N
DV-chlorophyll a DV-
H N H N N
chlorophyll b
CH3 CH3
H3C H3C
H H
H O H
O O
COOCH 3 O
COOCH 3
O CH3 O CH3
H3C H3C H H3C
H
CH3 H3C H3C H H3C
H
CH3
Molecule drawings:N. Montoya
 H2C
CH3
CH3
H3C
N N
Mg
N N
H3C CH3

H O
O COOCH 3
OH
chlorophyll c1
H2C H2C
CH3 COOCH 3
CH2 CH2
H3C H3C
N N
N N
Mg
Mg
N N N N
H3C CH3 H3C CH3
H O
O H O
COOCH 3
OH COOCH 3
OH
chlorophyll c2 chlorophyll c3
Molecule drawings:N. Montoya
Degradation by chemical processes:

Molecules become chemically and fotochemically

more labile in organic solvents
than in the cells
H2C Loss of metal
CH3
CH3
H3C Chla  Phaeophitin
N N
Mg
H N N  in organic solvents
CH3
H3C In dilute acids
H
H O  under high intensity of light
O
COOCH 3
O CH3
H3C H3C H H3C
H
CH3
 Degradation by chemical processes:

H2C
CH3
CH3
H3C
N N
Mg
H N N Epimerization
CH3 (HPLC: in SiO2):
H3C
Allomerization H
(oxidation by O2): H Cl  enolate  Cla’, b’
O
O
COOCH 3
O CH3
•Chl a  132 Hydroxiclhorophyll a H3C H3C H H3C
H
CH3
•Chl a  Cl a - Hyidroxilactone.

In alcoholic or hydro-alcoholic solutions Both processes

Specially in pH >7 can be minimized
by decreasing the temperature
 Degradation by chemical processes:

H2C
CH3
CH3
H3C
N N
Mg
H N N
CH3
H3C
H
H O
O
COOCH 3
O CH3

Loss of phytil group H3C H3C H H3C

H
CH3
Cl  chlorophyillide

In methanol or ethanol in basic medium

 Biodegradation:
Loss of metal:
Mg-dequelatase
To cyclic tetra-pirrols Formation of phaeophytins
H2C
perifercally modified CH3
CH3
(enzymatically, H3C
Specially in the absence of N N
light and O2): Mg Decarboximetilation
H N N Formation of
CH3 pirophaeophytins e
H3C pirophaeophorbides
Hydrolisis of the phytil H
ester H O
O
(chlorophyllase) COOCH 3
chlorophillide formation O CH3
H3C H3C H H3C
H
CH3

Allomerization
Epimerization (Chl-oxidase)
 Biodegradation:
To linear tetra H2C
pirrols
CH3
4
5 CH3
H3C
N N
Mg
Normally by oxidative opening
H N N
of the macrocycle ring, between
CH3
C-4 and C-5, H3C
H
C-5 stays as an aldehyde
H O
O
COOCH 3
O CH3
H3C H3C H H3C
H
CH3
 Carotenoids
Derive from carotene: C40H56

β- β- carotene

Isoprenoid
Polyen: units
Absorbtion
of light.
COLOUR
-carotene: ,-carotene
-carotene: ,-carotene
-carotene: ,-carotene
-carotene: ,-carotene
lycopene: ,-carotene
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
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: Phytoene
occurs in Dessaturation
thylakoid Lycopene
membranes Ciclization

,  -carotene ,  -carotene

Hydroxilation Hydroxilation
Zeaxanthin lutein
Deepoxidation
Light Dark Epoxidation
Anteraxanthin

Light Dark Epoxidation

Deepoxidation Can occur in the dark
Violaxanthin Depends a lot on light
VIOLAXANTHIN
CICLE
Rearrangement
Neoxanthin
 DIADINOXANTHIN CICLE

Diadinoxantin

epoxidation
+ 2H + O2 - H2O DARK LIGHT + 2H - H2O

Diatoxanthin
 Carotenoids

C40H56

β- β- carotene

Enzimatic Aldehydes,
hydroxilation Acetates ketones
Carboxi (OCOMe)
(CO2H), e lactones
carbometoxi Hydroxi-
Epoxidation
(CO2Me) carotenoids
ou metoxi as fatty acid esters,
(OMe) 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)

6
8
5

violaxanthin

6
8
5

neoxanthin
In basic medium:

In general stable

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

fucoxanthin
 Raphidophyceae
Distribution of chlorophylls among divisions/classes of phytoplankton

Chrysophyceae
Prymnesiophyceae
Dinophyta
Bacillariophyta
Eustimatophyta
Euglenophyta
Prasinophyceae
Chlorophyceae
Cryptophyta
Rhodophyta
Prochlorophyta
Cyanophyta

Tipo pyhtilat.
Division or

Pigment

DVChlb
MgDVP
DVchla
Chl c1
Chl c2
Chl c3
class

Chl a
Chlb

Chlc
 Raphidophyceae
Distribution of carotenes among divisions/classes of phytoplankton

Chrysophyceae
Prymnesiophyceae
Dinophyta
Bacillariophyta
Eustimatophyta
Euglenophyta
Prasinophyceae
Chlorophyceae
Cryptophyta
Rhodophyta
Prochlorophyta
Cyanophyta
Division or

Pigment
class

,
,
,
,

,
Distribution of xantophylls among divisions/classes of phytoplankton

Prochlorophyta

Rhodophyta

Cryptophyta

Chlorophyceae

Prasinophyceae

Euglenophyta

Eustimatophyta

Bacillariophyta

Dinophyta

Prymnesiophyceae

Chrysophyceae

Raphidophyceae
Division or class

Cyanophyta
/

Pigment

Aloxanthin
Anteraxanthin
Astaxanthin 2 2 2
19‘-Butanoil-
fucoxanthin
Cantaxanthin 2
Crocoxanthin
Diadinoxanthin
Diatoxanthin
Dinoxanthin
Echinenona 2 2
Fucoxanthin 1
 Distribution of xantophylls among divisions/classes of phytoplankton

Prochlorophyta

Rhodophyta

Cryptophyta

Chlorophyceae

Prasinophyceae

Euglenophyta

Eustimatophyta

Bacillariophyta

Dinophyta

Prymnesiop.

Chrysophyceae

Raphidophyceae
Division or

Cyanophyta
class

Pigment

19‘hexanoilfuco 1
Luteína
Monadoxanthin
Neoxanthin
P457+P468
Peridinina
Peridininol
Prasinoxanthin
Pirroxanthin
Sifonaxanthin 14 14
Sifoneina
Ést. Vaucheriax
Violaxanthin
Zeaxanthin
 Amphidinium carterae (Dinophyta)

Rzi =[lpigmi]/[chlorophyll a] Rz =[peridinin]/[chlorophyll a]

chlorophyll c2
chlorophyll a

dinoxanthin
peridinin
diadinoxanthin
Dunaliella tertiolecta (Chlorophyta)
Rzi =[lpigmi]/[chlorophyll a]
Rz =[lutein]/[chlorophyll a]

chlorophyll b

chlorophyll a

neoxanthin
violaxanthin lutein
anteraxanthin
 Hierarchical guide to the use of pigments

Pigment Significance
Chl a: an index of total algal biomass, excluding
prochlorophytes.
Unambiguous markers for algal types
DV-Chl a: an index of prochlorophyte biomass
DV-Chl b: unambiguous marker for prochlorophytes
Siphona xanthin esters: unambiguous marker for Type 2 prasinophytes
(Ege land et al., 1997)
Prasinoxanthi n: unambiguous marker for Type 3 prasinophytes
Peridinin : Type 1 dinoflagell ates
Alloxanthi n: Cryptophytes
Gyroxanthi n diester: Dinoflagell ates 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

Pe Rt Pigment Observed Published Reference

ak identification λmax λmax
no
.
(mi (nm) (nm)
n)
1 11. Erythroxanthin 465 469 Takaichi et al.
4 sulfate (1991)
2 18. Bacteriorubixa 513 510 Takaichi et al.
4 nthinal (1988)
3 19. Zeaxanthin (428), (428), Jeffrey et al.
1 Kobližek Arch Microbiol (2003)
Michal 454,180
482 454, 481
: 327–338 (1997)
4 20. Bacteriochloro 359, 580, 358, 577, Scheer (1991)
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

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 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 Fatores que afetam
(≤95 % dos compostos em poucos dias) a taxa
• digestão por herbívoros, de degradação:
• enzimática, na senescência celular
• oxidação química, microbiológica e
pela luz.
• Tempo para chegar
ao fundo
Nos sedimentos:
taxa de degradação menor, especialmente • Tipo de pigmento
em condições anóxicas. Depende de: • Grau de ataque
• intensidade de luz e da químico e biológico
• bioturvação invertebrada
 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: Chl a‘ and phaeophytin: Pirophaeophitins and steril
Degradation products due degradação products due to Chlorins: degradation
to zooplankton Environmental stress 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 Afinidade taxonômica
Funcionais
b,b-caroteno 0 Cianobactérias, algas eucarióticas e plantas vasculares
b,e-caroteno 0 Criptofitas
Aloxantina 2 Cryptofitas
Luteina 2 Clorófitas
Neoxantina 4 Clorófitas
Violaxantina 4 Chrisofitas e Clorófitas
Fucoxantina 5 Chrisofitas e Diatomáceas
Diatoxantina 2 Diatomáceas
Diadinoxantina 3 Dinoflagelados, Crisofitas e Diatomáceas
Peridinina 6 Dinoflagelados
Dinoxantina 4 Dinoflagelados
Zeaxantina 2 Cianobactérias, Clorófitas
Myxoxantofila 3 Cianobactérias
Echinenona 1 Cianobactérias e zooplâncton (Cladocera)
Cantaxantina 2 Cianobactérias e zooplâncton (Cladocera)
Astaxantina 4 Zooplâncton (Crustacea)
Okenona 2 Bactérias fotossintéticas (Chromatiaceae)
Scytonemina-1, -2 4 Organismos fotossintéticos expostos a alta radiação UV
Estáveis, abundantes (adaptado de Buchaca & Catalan 2008)
 Chlorophylls :

Pigmento Afinidades taxonômicas

Bacteriofeofitina-a Bactérias fotossintéticas (Rodospirillaceae e Chromatiaceae)
Bacterioclorofila-e Bactérias fotossintéticas (variedades marrons de Chlorobiaceae)
Clorofila-a Razão molar Cl-a/forbinas a como indicador de preservação
Chlorofilídeo-a Produto de degradação da Cl-a, abundante em Diatomáceas
Cl-a (alômero) Produto de degradação da Cl-a
Cl-a (epímero) Produto de degradação da Cl-a
Feofitina-a1, -a2 Produto de degradação da Cl-a (senescência)
Feoforbídeo-a1, -a2, Produto de degradação da Cl-a („grazing“)
-a3, -a30, -a4
Clorofila-b Clorófitas
Feofitina-b1, -b2 Produto de degradação da Cl-b
Clorofila-c1 Crisofitas e Diatomáceas
Clorofila-c2 Crisofitas, Diatomáceas, Criptofitas e Dinoflagelados
Clorofila-c3 Crisofitas e Diatomáceas

(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 III
0 0

Vibrational
fine
structure
 Molecular structure x spectroscopic properties

Chromophore (polyene chain): Lenght   

carotenoid Conjug.  max

db. bonds (hexane)
phytoene 3 276 286 297

-carotene 7 378 400 425

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
Britton, 1995, Carotenoids,
Carbonyl groups 3 vol, Birkhäuser
 Molecular environment x spectroscopic properties

Solvent Approx. bathochromic shift1

Hexane, light petroleum, 0
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, 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
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 (HPLC–DAD-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
Filtration Separation
GF/F 47mm
(HPLC)

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

Phytoplankton pigments
 Separation with C30 columns: Development of a
Fase estacionária: computer-assisted method (Software Dry Lab)
C30 (YMC, C30, 5µm,
polimérica
250x4,6 mm ID
c3
alo-, diato-xanthins DV, MV cl b
Fase móvel: c1c2 e luteína
DV, MV cl a
A:CH3OH:TBA (28 mM)
70:30 (v/v)
B: CH3CH2OH
pH 6,5
Gradiente:
chlorophylls:
Fig A:30-100 % B, 50 min

luteína
diatoxanthin
aloxanthin
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

Resolution:
Mistura-teste
otimization Van Heukelem e Thomas, Journal of Chromatography A, 910 (2001) 31-49
for chlorophylls
And for carotenoids in Resolution: separation mono/divynil clh a, b
Sparate runs They don‘t separate in C18 !! (depends on aliphatic chain?)
 Separation with C8 columns:
1) Development of a computer-assisted method (Software Dry Lab)

Zeaxanthin, luteína, DV, MV cl b

DV, MV cl a

c1 +clorofilídeo a
c3
Fase estacionária:

C2 +MgDVP
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

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

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

Fase móvel:
A: CH3OH : CH3CN : pirid.acet. (50:25:25); B: CH3OH : CH3CN : acetona (20:60:20)
 C8, Zapata R=0,8
cl b/DV cl b
Zeaxanthin, R< 0,5
dihidroluteína
Clor c2
R>1 4k Hex
/9‘cis Neo
MgDVP R> 1,25

R< 0,5 cl b/DV cl b

R=1 R= 0,8
C8, Van Heukelem
4k Hex
/9‘cis Neo
Não resolve

Pigment mixture, S. Wright, Course Notes

C8: better for chlorophyll c family
Comparison 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
 Separation of complex samples, method
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,
Método Airs et Al.
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
Extrato de amostra de sedimento (Priest Pot)
Airs et al.; Journal of Chromatography a 917 (2001) 167-177
 19,-butanoilfuco

zeaxanthin

Cl a + DV cla
Cl b + DV clb
diadinoxanthin

diatoxanthin
luteina
peridinina

dinoxanthin
aloxanthin
prasinoxanthin
neoxanthin

violaxanthin
Cl c2

fucoxanthin
Cl c3
Fase estacionária:
Spherisorb ODS1/ C18
250 x 4,6 mm – 5 m
Fase móvel:
A: CH3OH 0,3 M em Labor. UFF, Cromatógrafo Bischoffanalysentechn., Mistura-teste
(DHI), 100µL injetados na fase A,
NH4Ac : ACN : H20
(51:36:13)
B: AcetEtila: ACN (70:30)
Vazão: 1,2 ml/min Separates:,-carotene, ,-carotene, Aloxanthin,Lutein, Neoxanthin,
Violaxanthin, Fucoxanthin, Diatoxanthin,Diadinoxanthin, Peridinina,
Dinoxanthin, Zeaxanthin, Mixoxantophyll, Equinenone, Cantaxanthin,
Gradiente:0 a 25 % B em Astaxanthin, Okenone, Scytonemin-1, -2, Bacteriophaeophytin-a,
5 min, isocr.5 min, Bacteriochlorophyll-e, chlorophyll-a, Chlorophilide-a, Chl-a Allomer and Epimer,
25% a 100% B phaeophytin- a1, a2, phaeophorbide -a1, -a2, -a3, -a3’, -a4, chlorophyll b,
em 20 min. 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 = (Chl a)c1 + (Chl a)c2 + (Chl a)c3 + ...... + (Chl a)cn

Calculation of (Chl a)cn ?

Easy !
 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

Cianobactérias zeaxanthin Rzea/cla

Rlut/cla
Clorophyta luteína
Rper/cla
Dinophyta peridinina
Problem:
Ralo/cla Fixed R
Cryptophyta aloxanthin
not necessarily
.......................... ....................... ....................... Corresponds
Rfuco/cla To the ratios
Bacyllariophyta fucoxanthin
In the samples

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

{

Fixed (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 + .........+ 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
(Chl a)cn pigment
% of ech class
MÉTODO 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
 MATRIZ F: Razões Ri =[lpigmi]/[chlorophyll a para cada classe
PER BUT FUC HEX NEO PRA VI0L ALO LUT ZEA CLB CLA

Prasinophyt
a 0 0 0 0 0,061 0,127 0 0,004 0 0 0,381 0,403
Dinophyta 0,515 0 0 0 0 0 0 0 0 0 0 0,485

Cryptophyta 0 0 0 0 0 0 0 0,186 0 0 0 0,814

Haptophyta3 0 0 0 0,630 0 0 0 0 0 0 0 0,370
Haptophyta4 0 0,104 0,247 0,227 0 0 0 0 0 0 0 0,422
Chorophyta 0 0 0 0 0,040 0 0,035 0 0,127 0,006 0,165 0,628
Synecho. 0 0 0 0 0 0 0 0 0 0,258 0 0,742
Diatomaceas 0 0 0,430 0 0 0 00 0 0 0 0,570

MATRIZ S: experimental C: contribuição de cada

classe (a ser determinada)
Amostra 1: (Chl a)t1 (Zea)1 (Lut)1 ....... (Fuco)1
Amostra 2: (Chl a)t2 (Zea)2 (Lut)2 ....... (Fuco)2 Clpras
.................. ............ ......... ........ ....... ClDin
ClCryp
Amostra n: (Chl a)tn (Zea)n (Lut) ....... (Fuco)n
ClHapt3
ClHapt4
FxC=S ClChlor
ClSyn
ClDiatom
Para uma fatorização 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)
 Juturnaíba reservoir as a
study model
42°

23°
Rio de Janeiro
State

QuickTime™ and a
decompressor
are needed to see this picture.
Marcelo Marinho e Silvana V. Rodrigues
 OBJETIVOS

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
 METODOLOGIA
Fitoplâncton
–Coletas quinzenais - jun/96 - mai/97 (estação central)
–Biovolume
• método de sedimentação (Utermöhl, 1958)

Pigmentos

Amostra Injeção e análise

(0,25 - 1,8 L) HPLC

CONDIÇÕES
• Filtração (GF/C) CROMATOGRÁFICAS
• Congelamento
(CO2 sólido) • Coluna C18 - fase reversa
• Gradiente alta pressão
(modificado de Garrido & Zapata, 1993)
Extração • Detecção - 440nm
Metanol 100%
 Biomass (chlorophyll a)

Contribution calculated by marker pigments

Razão Xan/Chl-a CHEMTAX

300 300
a) a)
Cyanobacteria Cyanobacteria
250 250
Chlorophyceae Chlorophyceae
200 Cryptophyceae 200 Cryptophyceae

-1
µg L

µg L
150 Bacillariophyceae Bacillariophyceae
150
-1

Dinophyceae Dinophyceae
100 100
50 50
0 0
Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Aprb)May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Aprb)
May
100 1996 1997 100 1996 1997
relative contribution

relative contribution
80 80

60 60

40 40

20 20

0 0
Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Apr May Jun Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr Apr May
1996 1997 1996 1997
Biovolume
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)

 Biomass (Biovolume)
mg/L
90
cyanobacteria
diatoms
60 cryptomonads
dinoflagellates
green algae
30 others

0
Jun Jul Aug Sep Oct Nov Nov Dec Jan Feb Mar Apr May
1996 1997

Microcystis aeruginosa 20 mg/L

Anabaena spiroides
Cylindrospermopsis raciborskii

Percentages of phytoplankton assemblages as dominant groups

of species, by period in Juturnaíba 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 c lasses found by
pigment data and by biovolume calculation (significant *p
< 0.05, **p < 0. 01; n = 25).
Ratio Xan/Chl-a CHEMTAX
Dinophyceae 0.20 0.27
Bacillariophyceae 0.64* 0.76**
Cryptophyceae 0.39 0.73**
Chlorophyceae 0.39 -0.35
Cyanobacteria 0.89** 0.97**
Biovolume total 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.
 GUANABARA
12 SAMPLING SITES:
BAY
SAMPLING FREQUENCE:
- 12 CAMPAIGNS
- JANUARY TO AUGUST RJ/BRAZIL
(SUMMER/AUTUMN) 2006
HOMOGENEITY OF SAMPLES
WITHIN EACH DATA MATRIX

Data processing: 5 2
CHEMTAX:
Samples divided in 5 3
environmentally
different groups 4
1